Methods for enhancing antigen presentation

ABSTRACT

We describe (1) a method of enhancing antigen presentation, comprising the step of supplying to an antigen presenting cell such as a dendritic cell, or precursor cell, an inhibitor of Toll-related receptor (TRR) signalling and (2) a method of inhibiting antigen presentation, comprising the step of supplying to an antigen presenting cell such as a dendritic cell, or precursor cell, an enhancer of Toll-related receptor (TRR) signalling. The inhibitor of TRR signalling may be a dominant negative mutant of MyD88, for example MyD881pr.

This application is the US national phase of international applicationPCT/GB01/05724 filed 21 Dec. 2001, which designated the US.

The present invention relates to modulation of the immune system,particularly modulation of antigen presentation by dendritic cells.

Central to the recognition mechanisms of the immune system are a numberof germline-encoded receptors known as toll-like receptors (TLRs) (1).Individual TLRs activate specialised anti-fungal or anti-bacterial genesthrough the activation of the NF-κB transcription factors (2). Thus,TLR4 has been shown to confer responsiveness to bacteriallipopolysaccharide (3) whereas TLR2 confers responsiveness to bacterialpeptidoglycan and lipoteichoic acid as well as yeast carbohydrates (4).9 TLRs are currently known (8) and many more expected to exist.

Although the extracellular portions of Toll-related receptors (TRRs),including TLRs, are relatively divergent, the cytoplasmic portions aremore conserved. They contain a well-defined region known as the tolldomain, which is also found in the cytoplasmic portion of proteinscomprising the IL-1 receptor, the IL-18 receptor and other receptorsbroadly termed the IL-1 receptor family. In addition, solublecytoplasmic proteins such as MyD88, TollIP, Mal and TIRAP can have Tolldomains. TLRs and IL-1 receptor use an analogous framework ofsignalling; upon ligand binding, they recruit the adaptor molecule MyD88through homotypic interactions with a toll domain that MyD88 contains inits C-terminus. MyD88, in turn, recruits IRAK and TRAF-6 to activateNF-κB and mitogen-activated,protein kinases. TollIP may also be involved(2. Burns et al (2000) Nature cell Biol. 2, 346-351).

The MyD88 (myeloid differentiation protein) is considered to have amodular organisation consisting of an N-terminal death domain (DD)separated by a short linker from a C-terminal Toll domain (reviewed in(5)). The N-terminal DD is related to a motif of approximately 90 aminoacids that is considered to mediate protein-protein interactions withother DD sequences forming either homo- or heterodimers (Boldin et al(1995) J Biol Chem 270, 387-391).

The MyD88 Toll domain has about 130 amino acids (Mitcham et al (1996) JBiol Chem 199, 144-146). Toll domains are also considered to mediateprotein-protein interactions with other Toll domains forming eitherhomo- or heterodimers (see (5)).

DD and Toll-Toll interactions are considered to be involved in directingsignalling pathways. MyD88 is considered to bind via its Toll domain toTLRs and the IL-1 receptor (when bound to ligand). In turn, MyD88 isconsidered to bind via its DD to other DD-containing proteins; inparticular it is considered to bind to IRAK and TRAF-6, therebyactivating NF-κB and mitogen-activated protein kinases (2).

Antigen presenting cells are well known in the art and include dendriticcells (see Janeway, C A Jr & Tavers, P, Immunobiology (3rd Edition),Editions Current Biology/Churchill Livingstone and Garland Publishing).They are highly specialised cells that can process antigens and displaytheir peptide fragments on the cell surface, together with moleculesrequired for lymphocyte activation. The most potent antigen—presentingcells are dendritic cells, macrophages and B cells.

Dendritic cells (DC) are professional antigen-processing and -presentingcells which are critical to the development of primary MHC-restrictedT-cell immunity. They originate from a CD34⁺ precursor in bone marrow,but can also be derived from a post colony-forming unit CD14⁺intermediate in the peripheral blood. DC migrate to peripheral sites inskin, mucosa, spleen and thymus. They have been implicated in a varietyof clinically important processes, including allograft rejection, atopicdisorders, autoimmunity and anti-tumour immunity.

DC are cultured ex vivo from CD34⁺ stem cells or CD14⁺ peripheral bloodmonocytes using cytokines, principally GM-CSF, IL-4 and TNFα. DC fromboth these sources are immunocompetent and can take up exogenouslypresented antigen, process it and then present it to cytotoxic T-cells(Grabbe et al (1995) Immunology Today 16, 117-121; Girolomoni &Ricciardi-Castagnoli (1997) Immunology Today 18, 102-104). DC cantransfer antigen-specific tumour immunity generated in vivo (Kwak et al(1995) Lancet 345, 1016-1020) and autologous DC pulsed with tumourantigen ex vivo can induce a measurable anti-tumour effect (Hsu et al(1996) Nature Medicine 2, 52-58). DC can be effectively pulsed using acrude tumour membrane lysate, purified peptides or peptide fragments.The ex vivo expansion of autologous dendritic cells from patients,loading with a peptide antigen and reinfusion as adoptive immunotherapy,is described in, for example, WO/00/26249.

We surprisingly show that there is an inhibitory signal, specific forantigen presenting cells (APCs) such as dendritic cells and macrophages,that acts through MyD88. The inhibitory signal may involve one or moreTRRs.

TRRs include molecules such as TLRs, IL-1 receptor family membersincluding IL-1 receptor and IL-18 receptor and cytoplasmic proteins suchas MyD88, TollIP, Mal and TIRAP.

Molecules that modulate, preferably block TRR signalling in APCs, suchas dendritic cells, for example loss-of-function (inhibitory eg dominantnegative) forms of MyD88, may be used as immunostimulatory agents, forexample with DNA vaccines and cancer immunotherapy. For example,incorporation of inhibitory MyD88 into DNA vectors stimulates T cell andantibody responses more efficiently than if the vectors are used alone.

A first aspect of the invention provides a method of enhancing antigenpresentation, comprising the step of supplying to an antigen-presentingcell (APC), such as a dendritic cell, or precursor cell, a modulator,preferably an inhibitor, of a Toll-related receptor (TRR) signalling.The modulator activates APCs. for example DCs, for example enhancesantigen presentation by APCs, for example DCs.

A second aspect of the invention provides a method of inhibiting antigenpresentation, comprising the step of supplying to an antigen-presentingcell (APC), such as a dendritic cell, or precursor cell, a modulator,preferably an enhancer, of Toll-related receptor (TLR) signalling.

The inhibitor of Toll-related receptor (TRR) signalling preferablyinhibits a TRR signalling pathway found in APCs, such as dendriticcells, or a precursor thereof. In further preference, the inhibitorinhibits a TRR signalling pathway, the inhibition of which inducesactivation of immature dendritic cells and/or enhancement ofantigen-presenting function and may induce NF-κB nuclear translocationor the activation of MAP kinases. Thus, the TRR signalling pathway isconsidered to contribute to maintenance of immature APCs, such asdendritic cells, in the immature form, and to maintenance of NF-κB in aninactive form. Activation of the TRR signalling pathway may reduce theresponse of immature APCs, such as dendritic cells, to maturing factors,for example GM-CSF and IL4. ie may reduce the number of mature APCs,such as dendritic cells, formed, or may increase the time or dose ofmaturing factors needed for a given number of mature APCs, such asdendritic cells, to form. Activation of the TRR signalling pathway mayreduce the ability of mature APCs, such as dendritic cells, to induce aMLR (mixed lymphocyte reaction), a test of APC, such as dendritic cell,function well known to those skilled in the art. The APCs, such asdendritic cells, are incubated with allogeneic T cells and proliferationof the cells is measured, for example by measuring tritiated thymidineuptake after 6 days. For example, 10⁵ T cell may be plated with gradeddoses (for example from 50 to 10000 per well) of dendritic cells in a96-well round-bottom microtiter plate.

Typically, the APC is a professional antigen-presenting cell such as amucosal cell, macrophage or B cell. MHC Class II molecules are found inprofessional APCs. Professional APCs are characterised by the presenceof costimulatory molecules such as CD80 and CD86 as defined by Mellmanet al (1998) Trends Cell Biol. 8, 231-237.

Typically, isolated precursor or dendritic cells in which the pathway isstimulated express higher levels of HLA-DR, MHC Class I and CD80/86compared to unstimulated cells.

A list of DC surface markers regulated upon enhancement ofantigen-presenting function is given in Banchereau et al (2000) Ann.Rev. Immunol. Dendritic cell surface markers include high CCR1, CCR5.CCR6 but low CCR7 chemokine receptors; high CD68; low levels of MHCClass I (HLA-A, B, C) and MHC Class II (HLA-DR, HLA-DQ and HLA-DP); lowco-stimulatory molecules such as CD40, CD54, CD80. CD83 and CD86 and noDC-LAMP. Activated DC with increased antigen presentation have low CCR1,CCR5, CCR6; high CCR7; low CD68; high surface MHC Class I and II; highco-stimulatory molecules such as CD40, CD54, CD58, CD80, CD83, CD86;high DC-LAMP and high p55 fascin.

The term “inhibitor of Toll-related receptor (TRR) signalling” mayinclude a modulator of any one of the following steps in an APC (such asDC) TRR signalling pathway:

-   -   i) binding of an agonist or antagonist to a TRR, ie binding of a        TRR to an extracellular molecule,    -   ii) binding of the TRR to an intracellular molecule (TRR        interacting molecule), for example a polypeptide, for example an        adaptor molecule, for example a molecule comprising a Toll        domain, for example MyD88, Mal (Fitzgerald et al (2001) Nature        415, 78-83), TIRAP Horng et al (2001) Nature Immunol 7, 835-841)        or TollIP (Burns et al (2000) Nature Cell Biol 2, 346-351);    -   iii) binding of a TRR interacting molecule to a second        intracellular molecule, for example a polypeptide. For example        MyD88 may bind to a polypeptide comprising a death domain (DD),        as discussed above. A molecule that binds to MyD88 in an APC        such as a DC may be IRAK or TRAF-6 or possibly TollIP. IRAK is        described in Medzhitov et al (1998) Mol. Cell 2, 253-258; IRAK2        is described in Muzio et al (1997) Science 278, 1612-1615;        TollIP is describe in Burns et al (2000) Nature Cell Biol. 2,        346-351; and TRAF6 is described in Dushay & Eldon (1998) Am J        Hum. Genet. 62, 10-14 and Cao et al (1996) Nature 383, 443-446;    -   iv) further signalling steps (for example involving polypeptide:        polypeptide binding, or changes in phosphorylation state of a        polypeptide, or changes in particular phosphoinositide levels;        possibilities will be well known to those skilled in the art)        leading to inhibition of maturation of immature DC. The further        signalling steps may promote maintenance of NF-κB in an inactive        form. As an example of a further signalling step, the second        intracellular molecule may interact with a third or further        intracellular molecule, for example a polypeptide such as IκB        kinases or MAPkinases. The interaction may involve a change in        covalent modification of a polypeptide, for example        phosphorylation or dephosphorylation of the third intracellular        polypeptide by the second intracellular molecule or proteolytic        cleavage or ubiquitination.

As noted above, APCs such as dendritic cells are considered to have aTRR signalling pathway that inhibits NF-κB nuclear translocation(activation). Thus, it will be appreciated that a compound thatincreases NF-κB activation/nuclear translocation is not considered to bean activator of TRR signalling in an APC such as DC.

Similarly, a compound that inhibits NF-κB activation/nucleartranslocation is not considered to be an inhibitor of TRR signalling inan APC such as a DC.

It is preferred that the inhibitor or enhancer of TRR signalling in anAPC such as a DC does not act on a signalling pathway component or stepthat is shared by the APC inhibitory TRR signalling pathway and anysignalling pathway in an APC that leads to activation of NF-κB.

It is preferred that the inhibitor or enhancer of TRR signalling in anAPC acts at one of steps (i), (ii) or (iii) above, preferably step (ii)or (iii). Thus, it is preferred that the inhibitor or enhancer of TRRsignalling in an APC acts to modulate binding of an extracellularmolecule to a TRR (ie binding to an extracellular portion of the TRR);or to modulate binding of an intracellular molecule to a TRR (ie bindingto an intracellular portion of the TRR); or to modulate binding of suchan intracellular molecule to a further intracellular molecule. It isparticularly preferred that the inhibitor or enhancer of TRR signallingmodulates the binding of (functional) MyD88 to a TRR or the binding of(functional) MyD88 to a further intracellular molecule, for example apolypeptide comprising a DD. Thus, it is preferred that the inhibitor orenhancer of TRR signalling in an APC such as DC acts to modulatesignalling steps directly involving MyD88.

Alternatively, it is preferred that the modulator, for example inhibitoror enhancer, of TRR signalling in an APC such as DC acts to modulatesignalling steps directly involving another TRR interacting molecule oradapter, for example Mal (Fitzgerald et al (2001)) or TIRAP (Horng et al(2001)) or TollIP (Burns et al (2000)). TIRAP and Mal, for example, bothcontain TIR domains, but do not contain a Death Domain (DD). It isconsidered that wild-type TIRAP or wild-type Mal may have a similareffect in APC such as DC to a mutant of MyD88 which lacks a functionalDD, for example a similar effect to MyD88lpr. TollIP may contain a DD.It is considered that a mutant of TollIP which lacks a functional DD mayhave a similar effect in APC such as DC to a mutant of MyD88 which lacksa functional DD, for example a similar effect to MyD88lpr.

The modulator of TRR signalling in an APC such as DC may be TIRAP or Malor a fragment, fusion or variant thereof. Alternatively, it may beTollIP (Burns et al (2000)) or a fragment, fusion or variant thereof.

The inhibitor may also be a ribozyme which selectively destroy mRNAencoding a TRR, or an antisense molecule which prevents transcription ofa TRR.

Ribozymes which may be encoded in the genomes of the viruses orvirus-like particles herein disclosed are described in Cech andHerschlag, “Site-specific cleavage of single stranded DNA” U.S. Pat. No.5,180,818; Altman et al “Cleavage of targeted RNA by RNAse P” U.S. Pat.No. 5,168,053, Cantin et al “Ribozyme cleavage of HIV-1 RNA” U.S. Pat.No. 5,149,796; Cech et al “RNA ribozyme restriction endoribonucleasesand methods”, U.S. Pat. No. 5,116,742; Been et al “RNA ribozymepolymerases, dephosphorylases, restriction endonucleases and methods”,U.S. Pat. No. 5,093,246; and Been et al “RNA ribozyme polymerases,dephosphorylases, restriction endoribonucleases and methods; cleavessingle-stranded RNA at specific site by transesterification”, U.S. Pat.No. 4,987,071, all incorporated herein by reference.

Antisense oligonucleotides are single-stranded nucleic acids, which canspecifically bind to a complementary nucleic acid sequence. By bindingto the appropriate target sequence, an RNA-RNA, a DNA-DNA, or RNA-DNAduplex is formed. These nucleic acids are often termed “antisense”because they are complementary to the sense or coding strand of thegene. Recently, formation of a triple helix has proven possible wherethe oligonucleotide is bound to a DNA duplex. It was found thatoligonucleotides could recognise sequences in the major groove of theDNA double helix. A triple helix was formed thereby. This suggests thatit is possible to synthesise a sequence-specific molecules whichspecifically bind double-stranded DNA via recognition of major groovehydrogen binding sites.

By binding to the target nucleic acid, the above oligonucleotides caninhibit the function of the target nucleic acid. This could, forexample, be a result of blocking the transcription, processing,poly(A)addition, replication, translation, or promoting inhibitorymechanisms of the cells, such as promoting RNA degradations.

Antisense oligonucleotides are prepared in the laboratory and thenintroduced into cells, for example by microinjection or uptake from thecell culture medium into the cells, or they are expressed in cells aftertransfection with plasmids or retroviruses or other vectors carrying anantisense gene. Antisense oligonucleotides were first discovered toinhibit viral replication or expression in cell culture for Rous sarcomavirus, vesicular stomatitis virus, herpes simplex virus type 1, simianvirus and influenza virus. Since then, inhibition of mRNA translation byantisense oligonucleotides has been studied extensively in cell-freesystems including rabbit reticulocyte lysates and wheat germ extracts.Inhibition of viral function by antisense oligonucleotides has beendemonstrated in vitro using oligonucleotides which were complementary tothe AIDS HIV retrovirus RNA (Goodchild, J. 1988 “Inhibition of HumanImmunodeficiency Virus Replication by Antisense Oligodeoxynucleotides”,Proc. Natl. Acad. Sci. (USA) 85(15), 5507-11). The Goodchild studyshowed that oligonucleotides that were most effective were complementaryto the poly(A) signal; also effective were those targeted at the 5′ endof the RNA, particularly the cap and 5′ untranslated region, next to theprimer binding site and at the primer binding site. The cap, 5′untranslated region, and poly(A) signal lie within the sequence repeatedat the ends of retrovirus RNA (R region) and the oligonucleotidescomplementary to these may bind twice to the RNA.

Typically, antisense oligonucleotides are 15 to 35 bases in length. Forexample, 20-mer oligonucleotides have been shown to inhibit theexpression of the epidermal growth factor receptor mRNA (Witters et al,Breast Cancer Res Treat 53:41-50 (1999)) and 25-mer oligonucleotideshave been shown to decrease the expression of adrenocorticotropichormone by greater than 90% (Frankel et al, J Neurosurg 91:261-7(1999)). However, it is appreciated that it may be desirable to useoligonucleotides with lengths outside this range, for example 10, 11,12, 13, or 14 bases, or 36, 37, 38, 39 or 40 bases.

The anti-sense nucleic acid may be encoded by a suitable vector.

A further aspect of the invention provides a method of treating apatient in need of enhancement of antigen presentation comprising thestep of supplying a modulator, preferably an inhibitor, of Toll-relatedreceptor (TRR) signalling to the patient or to an antigen presentingcell such as a dendritic cell, or a precursor cell, of the patient. Themodulator activates APCs, such as DCs. The modulator may activatecertain signalling pathways, such as kinase pathways, for example MAPkinases p42 or p44/erk1/erk2, p54/Jnk, p38 MAP kinases or NFκB. Themodulator or inhibitor may be MyD88, Mal, TIRAP or TollIP or a fragment,fusion or variant any thereof, preferably a fragment, fusion or variantthat retains a functional TIR but may not have a functional DD (forexample may not have a DD, or may have a mutated DD which does notfunction as a DD, for example does not interact with another(functional) DD).

Thus, a further aspect of the invention provides a method of treating apatient in need of enhancement of antigen presentation comprising thestep of supplying MyD88, Mal, TIRAP or TollIP to the patient or to anantigen presenting cell such as a dendritic cell, or a precursor cell,of the patient. By MyD88, Mal, TIRAP or TollIP is included a fragment,fusion or variant thereof, preferably a fragment, fusion or variant thatretains a functional TIR but may not have a functional DD.

A further aspect of the invention provides a method of treating apatient in need of inhibition of antigen presentation comprising thestep of supplying a modulator, preferably an enhancer, of Toll-relatedreceptor (TRR) signalling to the patient or to an antigen-presentingdendritic cell, or precursor cell, of the patient. The modulator orenhancer may be a fragment, fusion or variant of MyD88, Mal or TIRAP orTollIP (or may be MyD88, Mal or TIRAP or TollIP), preferably a fragment,fusion or variant that retains some, but not all, of the binding orcatalytic properties of MyD88, Mal or TIRAP or TollIP, respectively. Themodulator or enhancer inhibits APCs, for example DCs, for exampleinhibits presentation of antigen by APCs, for example DCs. The modulatormay inhibit certain signalling pathways, such as kinase pathways, forexample MAP kinases p42 or p44/erk1/erk2, p54/Jnk, p38 MAP kinases orNFκB.

A further aspect of the invention provides a method of treating apatient in need of inhibition of antigen presentation comprising thestep of supplying a fragment, fusion or variant of MyD88, Mal, TIRAP orTollIP to the patient or to an antigen-presenting dendritic cell, orprecursor cell, of the patient. The fragment, fusion or variant inhibitsAPCs, for example DCs, for example inhibits antigen presentation byAPCs, preferably DCs. The fragment, fusion or variant preferably retainssome, but not all, of the binding or catalytic properties of MyD88, Mal,TIRAP or TollIP, respectively.

A further aspect of the invention provides a method of treating apatient in need of enhancement of antigen presentation comprising thestep of supplying to the patient, or to an antigen-presenting cell (APC)such as a dendritic cell, or dendritic precursor cell of the patient, aninhibitor of a signalling step (occuring in the APC such as a DC)directly involving MyD88. The inhibitor may be a dominant negativemutant of MyD88.

A further aspect of the invention provides a method of treating apatient in need of enhancement of antigen presentation comprising thestep of supplying to the patient, or to an antigen presenting cell suchas a dendritic cell, or precursor cell, of the patient, a dominantnegative mutant of MyD88.

Dominant negative mutants include portions of MyD88 that may includefragments of the molecule which include the death domain (DD) or Tolldomain, or portions thereof. Some examples are described in Reference(5). Preferably, the dominant negative mutants are ones which inhibitIL-1 or TRR signalling such as by the activation of IRAK or productionof cytokines in fibroblasts or HUVECs.

A further aspect of the invention provides a method of treating apatient in need of inhibition of antigen presentation comprising thestep of supplying to the patient, or to an antigen-presenting cell (APC)such as a dendritic cell, or precursor cell, of the patient, an enhancerof a signalling step (occuring in the APC such as DC) directly involvingMyD88. The enhancer may be functional MyD88, for example wildtype MyD88.

The signalling inhibitor or enhancer (for example TRR signallinginhibitor or enhancer, or inhibitor or enhancer of a signalling stepdirectly involving MyD88) is preferably specifically targeted to theAPCs, such as dendritic cells, or precursor cells, but this may not beessential.

The signalling inhibitor or enhancer may be supplied to the patient orpatient's cells by administration of the inhibitor or enhancer to thepatient or patient's cells, or by being expressed in cells of thepatient. Thus, supply may be achieved by administering to the patient(or to an APC or precursor cell of the patient; for example such a cellremoved from the patient for manipulation and subsequent return to thepatient) a recombinant polynucleotide encoding (and capable ofexpressing) a signalling inhibitor or enhancer.

The enhancer may increase MyD88 signalling activity. For example, theenhancer may be a MyD88 molecule with wild-type activity, or aconstitutively active MyD88 molecule. For example the enhancer may be aMyD88 molecule lacking a functional Toll domain (ie lacking a domainthat is capable of binding to a Toll domain); it may be a MyD88 mutantin which the Toll domain is deleted and/or non-functional, for example aMyD88-DD polypeptide as described in (5). Alternatively, the MyD88mutant may be one in which the death domain is deleted and/or isnon-functional, for example Myd88lpr. Thus, as an example, a recombinantpolynucleotide capable of expressing a wild-type MyD88 may beadministered to the patient.

The MyD88 molecule with wild-type activity may be wild-type MyD88.preferably a mouse human wild-type MyD88, still more preferably a MyD88that is expressed in mouse or human DC, or a mutated MyD88 which retainsthe activity of wild type MyD88 ie has a functional Toll domain and afunctional death domain (DD; ie has a domain that is capable of bindingto a death domain). It is preferred that the MyD88 Toll domain and/or DDare unmutated, ie that any mutation lies outside these domains.

It is preferred that the MyD88 has the sequence indicated in Hardiman etal (1996) Oncogene 13, 2467-2475; or Bonnert et al (1997) FEBS Lett.402, 81-84; or Hardiman et al (1997) Genomics 45, 332-339, all of whichare human. The human sequence is also given in Gen Bank Accession No.NM-002468.

The mouse MyD88 sequence is given in Harroch et al (1995) Nucl. AcidsRes. 23, 3539-3546 and Hardiman et al (1997) Genomics 45, 332-339 andGen Bank Accession No. NM-010851.

Human MyD88 is 82% identical in amino acid sequence to the mouse MyD88.

The human and mouse sequences for TIRAP are shown in Horng et al (2001)Nature Immunol 7, 835-841 and in Genbank Accession Nos AF378129 andAF378131, respectively.

The human and mouse sequences for Mal are shown in Fitzgerald et al(2001) Nature 413, 78-83.

The human, mouse and C. elegans sequences for TollIP are given in Burnset al (2000) Nature cell Biol 2, 346-351.

It is preferred that any mutation is a conservative substitution, aswell known to those skilled in the art. By “conservative substitutions”is intended combinations such as Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn,Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. Mutations may be made using themethods of protein engineering and site-directed mutagenesis as wellknown to those skilled in the art.

The three-letter and one-letter amino acid code of the IUPAC-IUBBiochemical Nomenclature Commission is used herein. The sequence ofpolypeptides are given N-terminal to C-terminal as is conventional. Itis preferred that the amino acids are L-amino acids, but they may beD-amino acid residues.

An enhancer of MyD88 signalling may act by binding to MyD88 or a bindingpartner of MyD88, for example by binding to a binding partner thatinhibits the signalling activity of MyD88. For example MyD88lpr may actby displacing a natural inhibitor of TRR signalling. The naturalinhibitor may be related to the SODD polypeptide that inhibits TNFαsignalling. SODD is described in Jiang et al (1999) Science 283,1852-1855.

The inhibitor may be an inhibitor of MyD88 signalling activity. It maybind to MyD88 or a binding partner of MyD88 and inhibit binding of MyD88to a binding partner. This may disrupt signalling via MyD88. Forexample, the inhibitor may inhibit binding of MyD88 to the Toll domainof a TRR, and/or may inhibit binding of MyD88 to a polypeptidecomprising a DD, for example IRAK or TRAF-6 or TollIP.

The inhibitor or enhancer may be a drug-like compound. The term“drug-like compound” is well known to those skilled in the art, and mayinclude the meaning of a compound that has characteristics that may makeit suitable for use in medicine, for example as the active ingredient ina medicament. Thus, for example, a drug-like compound may be a moleculethat may be synthesised by the techniques of organic chemistry, lesspreferably by techniques of molecular biology or biochemistry, and ispreferably a small molecule, which may be of less than 5000 daltons. Adrug-like compound may additionally exhibit features of selectiveinteraction with a particular protein or proteins and be bioavailableand/or able to penetrate cellular membranes, but it will be appreciatedthat these features are not essential.

The inhibitor or enhancer may be an antibody, by which term is includedantibody fragments or antibody-like molecules, as well known to thoseskilled in the art. Preferably, the antibody binds to MyD88 (or otheradapter molecule, for example Mal, TIRAP or TollIP) or to a bindingpartner of MyD88 (or other adapter molecule). For example, the antibodymay bind to the DD of MyD88 (and/or to the DD of a binding partner ofMyD88), and may disrupt binding of MyD88 to a DD of a binding partner ofMyD88. Alternatively, the antibody may bind to the Toll domain of MyD88(and/or to the Toll domain of a binding partner of MyD88), and maydisrupt binding of MyD88 to a Toll domain of a binding partner of MyD88.The antibody may preferably bind to an epitope of MyD88 that comprisesthe residue equivalent to Phe56 of wild-type mouse MyD88.

By an antibody is included an antibody or other immunoglobulin, or afragment or derivative thereof, as discussed further below.

The variable heavy (V_(H)) and variable light (V_(L)) domains of theantibody are involved in antigen recognition, a fact first recognised byearly protease digestion experiments. Further confirmation was found by“humanisation” of rodent antibodies. Variable domains of rodent originmay be fused to constant domains of human origin such that the resultantantibody retains the antigenic specificity of the rodent parentedantibody (Morrison et al (1984) Proc. Natl. Acad. Sci. USA 81,6851-6855).

That antigenic specificity is conferred by variable domains and isindependent of the constant domains is known from experiments involvingthe bacterial expression of antibody fragments, all containing one ormore variable domains. These molecules include Fab-like molecules(Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al(1988) Science 240, 1038); single-chain Fv (ScFv) molecules where theV_(H) and V_(L) partner domains are linked via a flexible oligopeptide(Bird et al (1988) Science 242, 493; Huston et al (1988) Proc. Natl.Acad. Sci. USA 85, 5879) and single domain antibodies (dAbs) comprisingisolated V domains (Ward et al (1989) Nature 341, 544). A gene: reviewof the techniques involved in the synthesis of antibody fragments whichretain their specific binding sites is to be found in Winter & Milstein(1991) Nature 349, 293-299.

By “ScFv molecules” we mean molecules wherein the V_(H) and V_(L)partner domains are linked via a flexible oligopeptide.

The advantages of using antibody fragments, rather than wholeantibodies, are several-fold. The smaller size of the fragments may leadto improved pharmacological properties. Effector functions of wholeantibodies, such as complement binding, are removed. Fab, Fv, ScFv anddAb antibody fragments can all be expressed in and secreted from E.coli, thus allowing the facile production of large amounts of the saidfragments. Fragments may also be expressed in cells of the patient.

Whole antibodies, and F(ab′)₂ fragments are “bivalent”. By “bivalent” wemean that the said antibodies and F(ab′)₂ fragments have two antigencombining sites. In contrast, Fab, Fv. ScFv and dAb fragments aremonovalent, having only one antigen combining sites.

Preferably, the antibody has an affinity for the epitope of betweenabout 10⁵.M⁻¹ to about 10¹².M⁻¹, more preferably at least 10⁸.M¹.

Antibodies reactive towards a chosen polypeptide may be made by methodswell known in the art. In particular, the antibodies may be polyclonalor monoclonal.

Suitable monoclonal antibodies to selected antigens may be prepared byknown techniques, for example those disclosed in “Monoclonal Antibodies:A manual of techniques”, H Zola (CRC Press, 1988) and in “MonoclonalHybridoma Antibodies: Techniques and Applications”, J G R Hurrell (CRCPress, 1982). Chimaeric antibodies are discussed by Neuberger et al(1988, 8th International Biotechnology Symposium Part 2, 792-799).Suitably prepared non-human antibodies can be “humanized” in known ways,for example by inserting the CDR regions of mouse antibodies into theframework of human antibodies.

Techniques for preparing antibodies are well known to those skilled inthe art, for example as described in Harlow, E D & Lane, D. “Antibodies:a laboratory manual” (1988) New York Cold Spring Harbor Laboratory.Suitable antibodies and techniques for preparing suitable antibodies maybe described in (5).

The antibody (particularly antibody fragment) may be joined to a moietythat facilitates uptake of the antibody by a cell, for example a DC. Forexample, the antibody may be linked to a lipophilic molecule orpolypeptide domain that is capable of promoting cellular uptake of themolecule or the interacting polypeptide, as known to those skilled inthe art. Thus, the moiety may derivable from the Antennapedia helix 3(Derossi et al (1998) Trends Cell Biol 8, 84-87), or from sequences ofHIV, generally tat, that permit entry into cells. Alternatively, apolynucleotide, for example cDNA, encoding the antibody may be deliveredin a vector, permitting expression of the antibody in the cell, asindicated above.

The inhibitor may be an inhibitory MyD88 molecule, preferably a dominantinhibitory MyD88 molecule (ie capable of inhibiting signalling bywild-type MyD88 molecules, for example in a cell in which wild-type andinhibitory MyD88 molecules are present). The inhibition may arise fromblocking interaction of endogenous wild-type MyD88 with a bindingpartner of the endogenous MyD88, for example a TRR.

The inhibitory MyD88 molecule may be a MyD88 molecule that is less ablethan MyD88, preferably substantially unable, to bind to a DD, forexample the DD of MyD88 or of IRK or TRAF-6. For example, the inhibitoryMyD88 may be less able than MyD88, preferably substantially unable, todimerise via the DD. The inhibitory MyD88 molecule may be a mutatedMyD88 molecule, for example a MyD88 molecule that is mutated in the DD,for example with a non-conservative mutation. For example, it may bemutated at the position equivalent to Phe56 of full length mouse MyD88,for example to Asn. It may be the mutated MyD88 molecule termed MyD88lpr(5) in which the N-terminal 53 amino acids of MyD88 are also absent.MyD88lpr has a point mutation (F56N; mouse sequence numbering) whencompared with wild-type MyD88, for example mouse wild-type MyD88. Thispoint mutation is in the DD and prevents dimerisation of the DD (5). Themutation corresponds to the lpr^(cp) mutation known to abolish cytotoxicsignalling of Fas, probably by disrupting the conformation of the DDdomain (Nagata (1994) Semin Immunol 6, 3-8; Huang, et al (1996) Nature384, 638-641).

The constructs for the wild-type MyD88 and dominant negative MyD88(MyD88-lpr) has been published (Burns K. et al. J Biol Chem 1998) butMyD88-lpr is wrongly described as a single amino acid mutation in itsdeath domain, where Phe⁵⁶ is mutated to Asn. This mutation correspondsto the lpr^(cp) mutation present in the death domain of Fas ligand whichabolishes its downstream signalling by disrupting the conformation ofthe death domain. Actually, in addition to the point mutation there is adeletion in its N-terminal domain of 53 amino acids (1-159 base pairs ofthe genebank sequence are missing). This deletion results in part of thedeath domain missing.

The inhibitory MyD88 may be a truncated version of MyD88, for example aMyD88 molecule in which all or part of the domain termed the DeathDomain is deleted. An inhibitory MyD88 molecule may be incapable or lesscapable of binding to a Death Domain than a wild-type MyD88 molecule.

It is preferred that the inhibitory MyD88 comprises a functional Tolldomain, ie a Toll domain that is capable of interacting with a Tolldomain, for example the Toll domain of a wild-type MyD88, for examplewild-type human or mouse MyD88 or a TRR. It is preferred that theinhibitory MyD88 comprises the full-length MyD88 Toll domain. Afull-length Toll domain may be necessary for Toll-Toll domaininteraction. Whilst not intending to be bound by theory, the inhibitoryMyD88 may then bind to the Toll domain of a Toll receptor molecule andthereby inhibit binding of a wild-type MyD88 to the Toll receptor,thereby inhibiting signalling from that receptor molecule.

Methods of measuring protein-protein interactions (and their enhancementor disruption) will be well known to those skilled in the art. Suitablemethods of measuring DD and Toll-Toll interactions are also described in(5). Suitable methods may include, for example, yeast two-hybridinteractions, co-purification, ELISA, co-immunoprecipitation,fluorescence resonance energy transfer (FRET) techniques and surfaceplasmon resonance methods. Thus, a MyD88 molecule may be consideredcapable of binding to or interacting with a DD or Toll domain if aninteraction may be detected between the said MyD88 polypeptide and apolypeptide comprising a DD or Toll domain by ELISA,co-immunoprecipitation or surface plasmon resonance methods or by ayeast two-hybrid interaction or copurification method. The preferredmethod is surface plasmon resonance.

The inhibitor or enhancer may be a peptidomimetic compound, for examplea peptidomimetic compound corresponding to a polypeptide inhibitor orenhancer discussed above.

The term “peptidomimetic” refers to a compound that mimics theconformation and desirable features of a particular peptide as atherapeutic agent, but that avoids potentially undesirable features. Forexample, morphine is a compound which can be orally administered, andwhich is a peptidomimetic of the peptide endorphin.

Therapeutic applications involving peptides may be limited, due to lackof oral bioavailability and to proteolytic degradation. Typically, forexample, peptides are rapidly degraded in vivo by exo- andendopeptidases, resulting in generally very short biological half-lives.Another deficiency of peptides as potential therapeutic agents is theirlack of bioavailability via oral administration. Degradation of thepeptides by proteolytic enzymes in the gastrointestinal tract is likelyto be an important contributing factor. The problem is, however, morecomplicated because it has been recognised that even small, cyclicpeptides which are not subject to rapid metabolite inactivationnevertheless exhibit poor oral bioavailability. This is likely to be dueto poor transport across the intestinal membrane and rapid clearancefrom the blood by hepatic extraction and subsequent excretion into theintestine. These observations suggest that multiple amide bonds mayinterfere with oral bioavailability. It is thought that the peptidebonds linking the amino acid residues in the peptide chain may breakapart when the peptide drug is orally administered.

There are a number of different approaches to the design and synthesisof peptidomimetics. In one approach, such as disclosed by Sherman andSpatola, J. Am. Chem. Soc., 112: 433 (1990), one or more amide bondshave been replaced in an essentially isoteric manner by a variety ofchemical functional groups. This stepwise approach has met with somesuccess in that active analogues have been obtained. In some instances,these analogues have been shown to possess longer biological half-livesthan their naturally-occurring counterparts. Nevertheless, this approachhas limitations. Successful replacement of more than one amide bond hasbeen rare. Consequently, the resulting analogues have remainedsusceptible to enzymatic inactivation elsewhere in the molecule. Whenreplacing the peptide bond it is preferred that the new linker moietyhas substantially the same charge distribution and substantially thesame planarity as a peptide bond.

Retro-inverso peptidomimetics, in which the peptide bonds are reversed,can be synthesised by methods known in the art, for example such asthose described in Mézière et al (1997) J Immunol. 159 3230-3237. Thisapproach involves making pseudopeptides containing changes involving thebackbone, and not the orientation of side chains. Retro-inversepeptides, which contain NH—CO bonds instead of CO—NH peptide bonds, aremuch more resistant to proteolysis.

In another approach, a variety of uncoded or modified amino acids suchas D-amino acids and N-methyl amino acids have been used to modifymammalian peptides. Alternatively, a presumed bioactive conformation hasbeen stabilised by a covalent modification, such as cyclisation or byincorporation of γ-lactam or other types of bridges. See, eg. Veber etal, Proc. Natl. Acad. Sci. USA, 75:2636 (1978) and Thursell et al,Biochem. Biophys. Res. Comm., 111:166 (1983).

A common theme among many of the synthetic strategies has been theintroduction of some cyclic moiety into a peptide-based framework. Thecyclic moiety restricts the conformational space of the peptidestructure and this frequently results in an increased affinity of thepeptide for a particular biological receptor. An added advantage of thisstrategy is that the introduction of a cyclic moiety into a peptide mayalso result in the peptide having a diminished sensitivity to cellularpeptidases.

One approach to the synthesis of cyclic stabilised peptidomimetics isring closing metathesis (RCM). This method involves steps ofsynthesising a peptide precursor and contacting it with a RCM catalystto yield a conformationally restricted peptide. Suitable peptideprecursors may contain two or more unsaturated C—C bonds. The method maybe carried out using solid-phase-peptide-synthesis techniques. In thisembodiment, the precursor, which is anchored to a solid support, iscontacted with a RCM catalyst and the product is then cleaved from thesolid support to yield a conformationally restricted peptide.

The methods of the invention may be used to treat any mammal such ashuman, dog, cat, horse, cow and the like. Preferably, the methods areused to treat a human patient.

A further aspect of the invention provides a method of identifying acompound which is an inhibitor of antigen presentation comprising thestep of identifying a compound that is capable of modulating, preferablyactivating TRR signalling, for example MyD88 signalling, in anantigen-presenting cell such as a dendritic cell, or precursor of anAPC. Preferably, modulation, for example activation, of TRR signallingmay be detected by observing a change in the interaction of MyD88 withan interacting molecule, for example polypeptide, for example anincrease of MyD88 binding to a TRR.

A further aspect of the invention provides a method of identifying acompound which is an enhancer of antigen presentation comprising thestep of identifying a compound that is capable of modulating, preferablyinhibiting TRR signalling, for example MyD88 signalling, (or enhancingMal, TIRAP or TollIP signalling) in an antigen-presenting cell, such asa dendritic cell, or precursor of an APC. The TRR signalling to beinhibited is signalling which inhibits DC maturation or function, asindicated above. Preferably, inhibition of TRR signalling may bedetected by observing a change in the interaction of MyD88 with aninteracting molecule, for example polypeptide, for example a decrease ofMyD88 binding to a TLR.

A change in IRAK or MAP kinase activity may be observed. IRAK activitymay be increased by MyD88lpr.

A further aspect of the invention provides a method of identifying acompound which is an inhibitor or enhancer of antigen presentation, forexample by an APC such as a DC, comprising the step of exposing a testcompound to MyD88 or to an APC binding partner of MyD88 (ie bindingpartner of MyD88 found in an APC such as a DC, or precursor thereof) anddetermining whether the compound is capable of binding to MyD88 or anAPC binding partner of MyD88. A compound capable of so binding isselected. Preferably the method further comprises the step ofdetermining whether the compound inhibits or promotes binding of MyD88to an APC binding partner of MyD88 and selecting a compound that iscapable of inhibiting or promoting such binding. The compound may befurther tested, for example by observing its effect on DC maturation orantigen presentation. The method may also comprise the step ofdetermining whether the compound binds to a mutant of MyD88, for exampleMyD88 lacking a functional Toll domain, or lacking a functional DD.

The binding partner may be a TRR or a polypeptide comprising a DD, asdiscussed above. Methods of identifying a MyD88 binding partner in anAPC such as a DC will be well known to those skilled in the art, andinclude co-precipitation and yeast two-hybrid methods and surfaceplasmon resonance and FRET.

A further aspect of the invention provides a method of identifying acompound which is an inhibitor or enhancer of antigen presentation, forexample by an APC such as a DC, comprising the step of exposing a testcompound to a TRR interacting molecule (adaptor molecule), for exampleMal or TIRAP or TollIP, or to an APC binding partner of such an adaptormolecule (ie binding partner of the adaptor molecule found in an APCsuch as a DC, or precursor thereof) and determining whether the compoundis capable of binding to the adaptor molecule (for example Mal or TIRAPor TollIP) or an APC binding partner of the adaptor molecule. A compoundcapable of so binding is selected. Preferably the method furthercomprises the step of determining whether the compound inhibits orpromotes binding of the adaptor molecule to an APC binding partner ofthe adaptor molecule and selecting a compound that is capable ofinhibiting or promoting such binding. The compound may be furthertested, for example by observing its effect on DC maturation or antigenpresentation. The method may also comprise the step of determiningwhether the compound binds to a mutant of the adaptor molecule, forexample a mutant of, for example, TIRAP or Mal or TollIP, lacking afunctional Toll domain, or lacking a functional DD.

A further aspect of the invention provides the use of MyD88 (or anotheradaptor molecule, for example TIRAP or Mal or TollIP) or an APC bindingpartner of MyD88 or other adaptor molecule in a method of identifying aninhibitor or enhancer of antigen presentation, for example by DC.

The compound may be a drug-like compound or lead compound for thedevelopment of a drug-like compound for each of the above methods ofidentifying a compound. It will be appreciated that the said methods maybe useful as screening assays in the development of pharmaceuticalcompounds or drugs, as well known to those skilled in the art.

The term “lead compound” is similarly well known to those skilled in theart, and may include the meaning that the compound, whilst not itselfsuitable for use as a drug (for example because it is only weakly potentagainst its intended target, non-selective in its action, unstable,difficult to synthesise or has poor bioavailability) may provide astarting-point for the design of other compounds that may have moredesirable characteristics.

A further aspect of the invention provides a compound identified oridentifiable by a screening method of the invention, ie as provided bythe preceding three aspects of the invention. A still further aspect ofthe invention provides a compound identified or identifiable by ascreening method of the invention for use in medicine. A further aspectof the invention provides an APC binding partner of MyD88 (or Mal orTIRAP or TollIP) or mutant thereof for use in medicine. A further aspectof the invention provides the use of a compound identified oridentifiable by a screening method of the invention, or an APC bindingpartner of MyD88 (or Mal or TIRAP or TollIP) or mutant thereof fortreating a patient in need of inhibition or enhancement of antigenpresentation, or in the manufacture of a medicamentfor treating apatient in need of inhibition or enhancement of antigen presentation.

Preferably the APC-binding partners of MyD88 (or Mal or TIRAP or TollIP)are DC-binding partners of MyD88.

A further aspect of the invention provides an inhibitor of Toll-relatedreceptor (TRR) signalling for use in medicine. A further aspect of theinvent ion provides an enhancer of Toll-related receptor (TRR)signalling for use in medicine, for example for treating a patient inneed of inhibition or enhancement of antigen presentation.

A further aspect of the invention provides an enhancer or inhibitor of asignalling step (occuring in an APC such as a DC) directly involvingMyD88 for use in medicine, for example for treating a patient in need ofinhibition or enhancement of antigen presentation.

Preferences for the said signalling inhibitors and enhancers are asindicated above. Preferably the signalling inhibitor is a dominantnegative mutant of MyD88, for example MyD88lpr or MyD88 lacking a deathdomain, or a compound that is capable of binding to MyD88, preferably asmall molecule or drug-like molecule, as discussed above. The signallinginhibitor or enhancer may also be TIRAP or Mal or TollIP or a fragment,fusion or variant thereof, as discussed above.

As indicated above, it will be appreciated that the signalling (forexample TRR or MyD88 signalling) inhibitor or enhancer (or compoundidentified or identifiable by a screening method of the invention) maybe supplied to the cell by means of expression (ie synthesis) of thesignalling inhibitor or enhancer (or compound) in the cell, for exampleexpression of the TRR signalling inhibitor or enhancer from arecombinant polynucleotide (ie a recombinant polynucleotide capable ofexpressing the TRR signalling inhibitor or enhancer) present in thecell. It will be appreciated that such supply by means of expression ofthe signalling inhibitor or enhancer or compound in the target cell maybe beneficial; for example, such supply may facilitate targeting of thesignalling inhibitor or enhancer to the desired cell. It may alsofacilitate temporally-extended presence of the signalling inhibitor orenhancer or the ability to supply the signalling inhibitor or enhancerto the cell.

It will be appreciated that it may be desirable to supply both amodulator, for example signalling inhibitor as defined above, (orcompound identified or identifiable by a screening method of theinvention which is an enhancer of antigen presentation) and an antigento the desired cell. It is preferred that either the signallinginhibitor/compound or antigen, preferably both, are supplied to thedesired cell by means of expression in the desired cell.

Similarly, it may be desirable to supply both a signalling enhancer (orcompound identified or identifiable by a screening method of theinvention which is an inhibitor of antigen presentation) and an antigento the desired cell. It is preferred that either the signallingenhancer/compound or antigen, preferably both, are supplied to thedesired cell by means of expression in the desired cell.

A further aspect of the invention provides a recombinant polynucleotideencoding a signalling inhibitor or enhancer (or an APC binding partnerof MyD88 or compound identified or identifiably by a screening method ofthe invention), for example a MyD88 polypeptide (for example aconstitutively active MyD88 polypeptide or dominant negative mutantMyD88 polypeptide) or Mal or TIRAP or TollIP polypeptide for use inmedicine. The signalling inhibitor or enhancer is preferably a dominantnegative MyD88 polypeptide as defined above. The recombinantpolynucleotide is preferably capable of expressing the signallinginhibitor or enhancer in an APC such as a dendritic cell, or an APCprecursor.

It is preferred that the recombinant polynucleotide encodes a modulatorof signalling pathways such as an inhibitor and an antigen that it isdesired to have presented by an APC. Thus, the recombinantpolynucleotide may comprise a portion encoding a signalling modulator,such as an inhibitor, and a portion encoding an antigenic molecule.Alternatively, the antigenic molecule may be encoded on a separatepolynucleotide molecule; this is less preferred. The signalling moleculeand antigen may be transcribed from a single promoter with an internalribosome entry site (IRES) for the second coding sequence.Alternatively, the signalling molecule and antigen may be transcribedfrom separate promoters.

The antigenic molecule may comprise more than one copy of one or moreepitopes. For example, it may comprise a single copy of a singleepitope-forming amino acid sequence, for example a sequence of betweenabout 8 and 30 amino acids, preferably about 10 to 18 amino acids, stillmore preferably about 15 amino acids in length. It may comprise multiplecopies of such an epitope-forming sequence, or single or multiple copiesof at least two different epitope-forming sequences. The antigenicsequences may be concatenated to form a domain-like structure, or may bedisposed at different points in a carrier polypeptide. Thepolynucleotide may encode one or several different antigenic moleculesor many, each of which may have one or more antigenic portions orepitopes.

The use of recombinant polyepitope vaccines for the delivery of multipleCD8 CTL epitopes is described in Thomson et al (1996) J. Immunol. 157,822-826 and WO 96/03144, both of which are incorporated herein byreference. In relation to the present invention, it may be desirable toinclude in a single vaccine, a peptide (or a nucleic acid encoding apeptide) wherein the peptide includes, in any order, one or moreantigenic amino acid sequences (for example each of between about 8 and18 amino acids in length), for example derived from a tumour-associatedantigen, and a CD4 T cell-stimulating epitope (such as from tetanustoxoid). Such “bead-on-a-string” vaccines are typically DNA vaccines.

For example, the antigenic molecule may comprise an epitope present ontransformed or cancerous cells (ie a tumour-associated antigen orepitope, for example the MAGE-1 antigen produced by a high proportion ofhuman melanoma tymours (van der Bruggen et al (1991) Science 254,1643)). Alternatively, it may comprise an epitope present on apathogenic organism, for example a virus, or on a cell (preferably ahuman cell) infected by a pathogenic organism, for example avirally-infected cell.

The epitope may be a T-cell epitope ie an epitope that is capable ofinducing a T-cell response (TH-1 response), eg. a T-helper cell responsepreferably a CD8+ cytotoxic T-cell response, but alternatively a CD4+helper T-cell response (TH-2 response) as well known to those skilled inthe art. A cytotoxic T-cell response may be undesirable in certaincases, for example when the antigen is a mycobacterial antigen (forexample Mycobacterium tuberculosis or M. leprae antigen).

A signalling enhancer as herein described may be capable of modulatingthe immune response to an antigen such that the TH1/TH2 balance of theimmune response is altered, preferably becoming more TH1 and less TH2. ATH2 response may be associated with allergy.

It is preferred if the cancer antigen is, or has at least one epitopepresent in, any of the following:

-   -   i) normal cellular proteins that are expressed at abnormally        high levels in tumours; eg cyclin D1 in a variety of tumours;        cyclin E in breast cancer; mdm 2 in a variety of tumours; EGF-R,        erb-B2, erb-B3, FGF-R, insulin-like growth factor receptor, Met,        myc, p53 and BCL-2 are all expressed in various tumours.    -   ii) normal cellular proteins that are mutated in tumours; eg Ras        mutations in a variety of tumours; p53 mutations in a variety of        tumours; BCR/ABL translocation in CML and ALL; CSF-1 receptor        mutations in AML and MDS; APC mutations in colon cancer; RET        mutations in MEN2A, 2B and FMTC; EGFR mutations in gliomas;        PML/RARA translocation in PML; E2A-PBX1 translocation in pre B        leukaemias and in childhood acute leukaemias.    -   iii) virally encoded proteins in tumours associated with viral        infection; eg human papilloma virus proteins in cervical cancer;        Epstein-Barr virus proteins in B cell lymphomas and Hodgkin's        lymphoma; HTLV-1 proteins in adult T cell leukaemia; hepatitis B        and C virus proteins in hepatocellular carcinoma; herpes-like        virus proteins in Kaposi's sarcoma.    -   iv) HIV encoded proteins in HIV infected patients.

Thus, the above cancer-associated antigens can be divided into threemain categories: (i) normal self antigens expressed at high levels intumour cells; (ii) mutated self antigens expressed in tumour cells;(iii) viral antigens expressed in tumours associated with viralinfection. Category (i) is preferred.

Three subtypes are included in category (i):

-   -   a) normal cellular proteins that are overexpressed;    -   b) proteins that are expressed in a tissue-specific fashion in        normal cells but also in tumours; and    -   c) proteins that are embryonic antigens, silent in most adult        tissues but aberrantly expressed in tumours.

Examples of b) and c) are:

-   -   b) tissue-specific differentiation antigens as targets for        tumour-reactive CTL such as GATA-1, IKAROS, SCL (expressed in        the haematopoietic lineage and in leukaemias); and        immunoglobulin constant regions (for treatment of multiple        myeloma); and    -   c) Wilms-tumour antigen 1 (WT1) for treatment of leukaemias and        Wilms tumour and carcinoembryonic antigens (CEA a foetal        protein) for liver and intestinal tumours.

In one embodiment, the cancer-associated antigen may be provided by acrude extract of a tumour sample.

Overexpression of oncogene-encoded proteins in human tumours and mutatedoncogenes expressed in human tumours are described in Stauss & Dahl(1995) Tumour Immunology, Dalgleish/Browning, Chapter 7. incorporatedherein by reference.

Thus, it is preferred if the patient to be treated has cancer; morepreferably any one of breast cancer; bladder cancer; lung cancer;prostate cancer; thyroid cancer; leukaemias and lymphomas such as CML,ALL, AML PML; colon cancer; glioma; seminoma; liver cancer; pancreaticcancer; bladder cancer; renal cancer; cervical cancer; testicularcancer; head and neck cancer; ovarian cancer; neuroblastoma andmelanoma.

CML is chronic myelocytic leukaemia; ALL is acute lymphoblasticleukaemia; AML is acute myelocytic leukaemia; and PML is pro-myelocyticleukaemia.

Alternatively, the patient may have or be at risk of any disease causedby a pathogen, particularly a bacterium, yeast, virus, trypanosome andthe like. It is preferred if the disease is caused by a chronicinfection with a pathogen. It is also preferred if the pathogen is onewhich is not readily cleared by the host immune system.

It is preferred if the disease is a viral infection; more preferably adisease caused by any one of HIV, papilloma virus, Epstein-Barr virus,HTLV-1, hepatitis B virus, hepatitis C virus, herpes virus or any virusthat causes chronic infection. It is particularly preferred if the virusis HIV.

Abnormally elevated amounts of a hormone produced by cells occur in somediseases such as certain types of thyroid disease. Thus, the method ofthe invention may be used to promote ablation of cells producing theelevated amounts of the hormone. The antigen may be the hormone thebiosynthetic enzymes involved in synthesis of the hormone, which may beoverproduced by the cell.

Patients with a bacterial infection, particularly an infection thatcauses chronic infection may also be usefully treated. The bacterialinfection may be an intracellular infection. Thus, the method may beuseful in treating tuberculosis.

The method may also be used to treat malaria.

Other patients who may benefit from enhancement or stimulation ofantigens presentation include those with prion-related diseases such asspongiform encephalopathies. The method of enhancing or stimulatingantigen presentation may be used to treat (including prophylactically)diseases or conditions characterised by aberrant types and/or abberantlyhigh levels of (harmful) molecules, for example polypeptides, in thebody, for example, levels of inflammatory mediators (for example,cytokines) associated with chronic inflammation; breakdown products ofcells or connective tissue matrix, for example fibronectrin fragments;β-amyloid polypeptide (associated with Alzheimer's disease). Stimulatingan immune response against such molecules may aid removal of themolecules from the body, thereby helping in resolution or prevention ofthe condition.

A patient in need of inhibition of antigen presentation may be a patientwith or at risk of an autoimmune disease or allergy, or a patient inreceipt of, or intended to be in receipt of, transplanted tissue, ie apatient in which inhibition of rejection of the transplanted tissue isrequired. Appropriate antigens will be known to those skilled in theart.

Autoimmune diseases that may be treated by this method of the inventioninclude rheumatoid arthritis, type I diabetes, multiple sclerosis,Crohn's disease, Hashimoto's thyroiditis, coeliac disease, myastheniagravis, pemphigus vulgaris, systemic lupus erythromatosus, and Gravesdisease. Allergies which may be treatable by the method described hereininclude allergies to the following allergens: Fel d 1 (the feline skinand salivary gland allergen of the domestic cat Felis domesticus—theamino acid sequence of which is disclosed in WO 91/06571), Der p I, Derp II, Der FI or Der fII (the major protein allergens from the house dustmite dermatophagoides—amino acid sequences disclosed in WO 94/24281).

The invention is applicable substantially to any allergy, includingthose caused by allergens present in any of the following: grass, treeand weed (including ragweed) pollens; fungi and moulds; foods eg fish,shellfish, crab lobster, peanuts, nuts, wheat gluten, eggs and milk;stinging insects eg bee, wasp and hornet and the chironomidae(non-biting midges); spiders and mites, including the house dust mite;allergens found in the dander, urine, saliva, blood or other bodilyfluid of mammals such as cat, dog, cows, pigs, sheep, horse, rabbit,rat, guinea pig, mouse and gerbil; airbourne particulates in general;latex; and protein detergent additives.

Allergies to proteins from the following insects may also be treated:housefly, fruit fly, sheep blow fly, screw worm fly, grain weevil,silkworm, honeybee, non-biting midge larvae, bee moth larvae mealworm,cockroach and larvae of Tenibrio molitor beetle.

A further aspect of the invention comprises a kit of parts, compositionor a chimaeric molecule, for example chimaeric polypeptide, comprising asignalling inhibiting or enhancing portion (which may be apolynucleotide encoding an antigenic molecule) and an antigenic portion(which may be a polynucleotide encoding an antigenic molecule), asdefined above. Either or both portions may further comprise atranslocating portion and/or a cell binding portion. The cell bindingportion is preferably capable of binding to a dendritic cell orprecursor thereof. The translocating portion may aid in internalisationof the molecule or at least the antigenic portion and preferably thesignalling inhibiting or enhancing portion. Thus, exogenously appliedpeptides may be linked to a HIV tat peptide. This may direct them intothe MHC Class I pathway for presentation by CTL (see, for example, Kimet al (1997) J. Immunol. 159, 1666-1668. Chimaeric molecules which maybe adapted in accordance with the present invention are described inWO95/31483.

Dendritic cells may be characterised by expression of the. CD80, CD86.CD40, CD1a, HLA-DR and/or CD83 cell surface molecules. Immaturedendritic cells may be CD34⁺ or CD14⁺. Thus, the cell binding portionmay be capable of binding to one or more of these cell surface molecules(for example, an antibody capable of binding to such a molecule). CD1aand CD83 are preferred for targeting DCs.

Polypeptides in which one or more of the amino acid residues arechemically modified, before or after the polypeptide is synthesised, maybe used as antigen providing that the function of the polypeptide,namely the production of a specific immune response in vivo, remainssubstantially unchanged. Such modifications include forming salts withacids or bases, especially physiologically acceptable organic orinorganic acids and bases, forming an ester or amide of a terminalcarboxyl group, and attaching amino acid protecting groups such asN-t-butoxycarbonyl. Such modifications may protect the polypeptide fromin vivo metabolism.

The epitope(s) (for example epitope-forming amino acid sequences) may bepresent as single copies or as multiples, for example tandem repeats.Such tandem or multiple repeats may be sufficiently antigenic themselvesto obviate the use of a carrier. It may be advantageous for thepolypeptide to be formed as a loop, with the N-terminal and C-terminalends joined together, or to add one or more Cys residues to an end toincrease antigenicity and/or to allow disulphide bonds to be formed. Ifthe epitope, for example epitope-forming amino acid sequence, iscovalently linked to a carrier, preferably a polypeptide, then thearrangement is preferably such that the epitope-forming amino acidsequence forms a loop.

According to current immunological theories, a carrier function shouldbe present in any immunogenic formulation in order to stimulate, orenhance stimulation of, the immune system. The epitope(s) as definedabove in relation to the preceding aspects of the invention may beassociated, for example by cross-linking, with a separate carrier, suchas serum albumins, myoglobins, bacterial toxoids and keyhole limpethaemocyanin. More recently developed carriers which induce T-cell helpin the immune response include the hepatitis-B core antigen (also calledthe nucleocapsid protein), presumed T-cell epitopes such asThr-Ala-Ser-Gly-Val-Ala-Glu-Thr-Thr-Asn-Cys, beta-galactosidase and the163-171 peptide of interleukin-1. The latter compound may variously beregarded as,a carrier or as an adjuvant or as both.

Alternatively, several copies of the same or different epitope may becross-linked to one another; in this situation there is no separatecarrier as such, but a carrier function may be provided by suchcross-linking. Suitable cross-linking agents include those listed assuch in the Sigra and Pierce catalogues, for example glutaraldehyde,carbodiimide and succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate, the latter agentexploiting the —SH group on the C-terminal cysteine residue (ifpresent). Any of the conventional ways of cross-linking polypeptides maybe used, such as those generally described in O'Sullivan et al Anal.Biochem. (1979) 100, 100-108. For example, the first portion may beenriched with thiol groups and the second portion reacted with abifunctional agent capable of reacting with those thiol groups, forexample the N-hydroxysuccinimide ester of iodoacetic acid (NHIA) orN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), aheterobifunctional cross-linking agent which incorporates a disulphidebridge between the conjugated species. Amide and thioether bonds, forexample achieved with m-maleimidobenzoyl-N-hydroxysuccinimide ester, aregenerally more stable in vivo than disulphide bonds.

Further useful cross-linking agents include S-acetylthioglycolic acidN-hydroxysuccininide ester (SATA) which is a thiolating reagent forprimary amines which allows deprotection of the sulphydryl group undermild conditions (Julian et al (1983) Anal. Biochem. 132, 68),dimethylsuberimidate dihydrochloride and N,N′-o-phenylenedimaleimide.

If the polypeptide is prepared by expression of a suitable nucleotidesequence in a suitable host, then it may be advantageous to express thepolypeptide as a fusion product with a peptide sequence which acts as acarrier. Kabigen's “Ecosec” system is an example of such an arrangement.

Epitopes from different biological sources (for example differentpathogenic organisms) may be linked to other antigens to provide a dualeffect.

By epitopes is included mimotopes, as well known to those skilled in theart.

Suitable vectors or constructs which may be used to prepare a suitablerecombinant polypeptide or polynucleotide will be known to those skilledin the art.

A polynucleotide capable of expressing the required polypeptide orpolypeptides may be prepared using techniques well known to thoseskilled in the art.

Preferably, the polynucleotide is capable of expressing thepolypeptide(s) in the patient. The polypeptide(s), for example TRRsignalling inhibitor or enhancer, or antigen, as appropriate, may beexpressed from any suitable polynucleotide (genetic construct) as isdescribed below and delivered to the patient. Typically, the geneticconstruct which expresses the polypeptide comprises the said polypeptidecoding sequence operatively linked to a promoter which can express thetranscribed polynucleotide (eg mRNA) molecule in a cell of the patient,which may be translated to synthesise the said polypeptide. Suitablepromoters will be known to those skilled in the art, and may includepromoters for ubiquitously expressed, for example housekeeping genes orfor tissue-selective genes, depending upon where it is desired toexpress the said polypeptide (for example, in dendritic cells orprecursors thereof). Preferably, a dendritic cell or dendritic precursorcell-selective promoter is used, but this is not essential, particularlyif delivery or uptake of the polynucleotide is targeted to the selectedcells, eg dendritic cells or precursors. Dendritic cell-selectivepromoters may include the CD83 or CD36 promoters.

The nucleic acid sequence capable of expressing the polypeptide(s) ispreferably operatively linked to regulatory elements necessary forexpression of said sequence.

“Operatively linked” refers to juxtaposition such that the normalfunction of the components can be performed. Thus, a coding sequence“operatively linked” to regulatory elements refers to a configurationwherein the nucleic acid sequence encoding the inhibitor (or inducer,which is useful as described in more detail below), of NF-κB can beexpressed under the control of the regulatory sequences.

“Regulatory sequences” refers to nucleic acid sequences necessary forthe expression of an operatively linked coding sequence in a particularhost organism. For example, the regulatory sequences which are suitablefor eukaryotic cells are promoters, polyadenylation signals, andenhancers.

“Vectors” means a DNA molecule comprising a single strand, doublestrand, circular or supercoiled DNA. Suitable vectors includeretrovinises, adenoviruses, adeno-associated viruses, pox viruses andbacterial plasmids. Retroviral vectors are retroviruses that replicateby randomly integrating their genome into that of the host. Suitableretroviral vectors are described in WO 92/07573.

Adenovirus is a linear double-standard DNA Virus. Suitable adenoviralvectors are described in Rosenfeld et al, Science, 1991, Vol. 252, page432.

Adeno-associated viruses (AAV) belong to the parvo virus family andconsist of a single strand DNA or about 4-6 KB.

Pox viral vectors are large viruses and have several sites in whichgenes can be inserted. They are thermostable and can be stored at roomtemperature. Safety studies indicate that pox viral vectors arereplication-defective and cannot be transmitted from host to host or tothe environment.

Targeting the vaccine to specific cell populations, for example antigenpresenting cells, may be achieved, for example, either by the site ofinjection, use of targeting vectors and delivery systems, or selectivepurification of such a cell population from the patient and ex vivoadministration of the peptide or nucleic acid (for example dendriticcells may be sorted as described in Zhou et al (1995) Blood 86,3295-3301; Roth et al (1996) Scand. J. Immunology 43, 646-651). Inaddition, targeting vectors may comprise a tissue- or tumour-selectivepromoter which directs expression of the antigen at a suitable place.

Although the genetic construct can be DNA or RNA it is preferred if itis DNA.

Preferably, the genetic construct is adapted for delivery to a humancell.

Means and methods of introducing a genetic construct into a cell in orremoved from an animal body are known in the art. For example, theconstructs of the invention may be introduced into the cells by anyconvenient method, for example methods involving retroviruses, so thatthe construct is inserted into the genome of the (dividing) cell.Targeted retroviruses are available for use in the invention; forexample, sequences conferring specific binding affinities may beengineered into pre-existing viral env genes (see Miller & Vile (1995)Faseb J. 9, 190-199 for a review of this and other targeted vectors forgene therapy).

Preferred retroviral vectors are lentiviral vectors such as thosedescribed in Verma & Somia (1997) Nature 389, 239-242.

It will be appreciated that retroviral methods, such as those describedbelow, may only be suitable when the cell is a dividing cell. Forexample, in Kuriyama et al (1991) Cell Struc. and Func. 16, 503-510purified retroviruses are administered. Retroviral DNA constructs whichencode the desired polypeptide(s) may be made using methods well knownin the art. To produce active retrovirus from such a construct it isusual to use an ecotropic psi2 packaging cell line grown in Dulbecco'smodified Eagle's medium (DMEM) containing 10% foetal calf serum (FCS).Transfection of the cell line is conveniently by calcium phosphateco-precipitation, and stable transformants are selected by addition ofG418 to a final concentration of 1 mg/ml (assuming the retroviralconstruct contains a neo® gene). Independent colonies are isolated andexpanded and the culture supernatant removed, filtered through a 0.45 μmpore-size filter and stored at −70° C. For the introduction of theretrovirus into the target cells, it is convenient to inject directlyretroviral supernatant to which 10 μg/ml Polybrene has been added. Theinjection may be made into the area in which the target cells arepresent, for example subcutaneously.

Other methods involve simple delivery of the construct into the cell forexpression therein either for a limited time or, following integrationinto the genome, for a longer time. An example of the latter approachincludes liposomes (Nässander et al (1992) Cancer Res. 52, 646-653).Other methods of delivery include adenoviruses carrying external DNA viaan antibody-polylysine bridge (see Curiel Prog. Med. Virol. 40, 1-18)and transferrin-polycation conjugates as carriers (Wagner et al (1990)Proc. Natl. Acad. Sci. USA 87, 3410-3414). In the first of these methodsa polycation-antibody complex is formed with the DNA construct or othergenetic construct of the invention, wherein the antibody is specific foreither wild-type adenovirus or a variant adenovirus in which a newepitope has been introduced which binds the antibody. The polycationmoiety binds the DNA via electrostatic interactions with the phosphatebackbone. The adenovirus, because it contains unaltered fibre and pentonproteins, is internalised into the cell and carries into the cell withit the DNA construct of the invention. It is preferred if the polycationis polylysine.

Bacterial delivery is described in Dietrich (2000) Antisense NucleicAcid Drug Delivery 10, 391-399.

The DNA may also be delivered by adenovirus wherein it is present withinthe adenovirus particle, for example, as described below.

In the second of these methods, a high-efficiency nucleic acid deliverysystem that uses receptor-mediated endocytosis to carry DNAmacromolecules into cells is employed. This is accomplished byconjugating the iron-transport protein transferrin to polycations thatbind nucleic acids. Human transferrin, or the chicken homologueconalbumin, or combinations thereof is covalently linked to the smallDNA-binding protein protamine or to polylysines of various sizes througha disulfide linkage. These modified transferrin molecules maintain theirability to bind their cognate receptor and to mediate efficient irontransport into the cell. The transferrin-polycation molecules formelectrophoretically stable complexes with DNA constructs or othergenetic constructs of the invention independent of nucleic acid size(from short oligonucleotides to DNA of 21 kilobase pairs). Whencomplexes of transferrin-polycation and the DNA constructs or othergenetic constructs of the invention are supplied to the target cells, ahigh level of expression from the construct in the cells is expected.

High-efficiency receptor-mediated delivery of the DNA constructs orother genetic constructs of the invention using the endosome-disruptionactivity of defective or chemically inactivated adenovirus particlesproduced by the methods of Cotten et al (1992) Proc. Natl. Acad. Sci.USA 89, 6094-6098 may also be used. This approach appears to rely on thefact that adenoviruses are adapted to allow release of their DNA from anendosome without passage through the lysosome, and in the presence of,for example transferrin linked to the DNA construct or other geneticconstruct of the invention, the construct is taken up by the cell by thesame route as the adenovirus particle.

This approach has the advantages that there is no need to use complexretroviral constructs; there is no permanent modification of the genomeas occurs with retroviral infection; and the targeted expression systemis coupled with a targeted delivery system, thus reducing toxicity toother cell types.

“Naked DNA” and DNA complexed with cationic and neutral lipids may alsobe useful in introducing the DNA of the invention into cells of thepatient to be treated. Non-viral approaches to gene therapy aredescribed in Ledley (1995) Human Gene Therapy 6, 1129-1144. Alternativetargeted delivery systems are also known such as the modified adenovirussystem described in WO 94/10323 wherein, typically, the DNA is carriedwithin the adenovirus, or adenovirus-like, particle. Michael et al(1995) Gene Therapy 2, 660-668 describes modification of adenovirus toadd a cell-selective moiety into a fibre protein. Mutant adenoviruseswhich replicate selectively in p53-deficient human tumour cells, such asthose described in Bischoff et al (1996) Science 274, 373-376 are alsouseful for delivering the genetic construct of the invention to a cell.Thus, it will be appreciated that a further aspect of the inventionprovides a virus or virus-like particle comprising a genetic constructof the invention. Other suitable viruses or virus-like particles includeHSV, AAV, vaccinia, lentivirus and parvovirus.

Immunoliposomes (antibody-directed liposomes) are especially useful intargeting to cell types which over-express a cell surface protein forwhich antibodies are available, as is possible with dendritic cells orprecursors, for example using antibodies to CD1, CD14 or CD83 (or otherdendritic cell or precursor cell surface molecule, as indicated above).For the preparation of immuno-liposomes MPB-PE(N-[4-(p-maleimidophenyl)butyryl]-phosphatidylethanolamine) issynthesised according to the method of Martin & Papahadjopoulos (1982)J. Biol. Chem. 257, 286-288. MPB-PE is incorporated into the liposomalbilayers to allow a covalent coupling of the antibody, or fragmentthereof, to the liposomal surface. The liposome is conveniently loadedwith the DNA or other genetic construct of the invention for delivery tothe target cells, for example, by forming the said liposomes in asolution of the DNA or other genetic construct, followed by sequentialextrusion through polycarbonate membrane filters with 0.6 μm and 0.2 μmpore size under nitrogen pressures up to 0.8 MPa. After extrusion,entrapped DNA construct is separated from free DNA construct byultracentrifugation at 80 000×g for 45 min. Freshly preparedMPB-PE-liposomes in deoxygenated buffer are mixed with freshly preparedantibody (or fragment thereof) and the coupling reactions are carriedout in a nitrogen atmosphere at 4° C. under constant end over endrotation overnight. The immunoliposomes are separated from unconjugatedantibodies by ultracentrifugation at 80 000×g for 45 min.Immunoliposomes may be injected, for example intraperitoneally ordirectly into a site where the target cells are present, for examplesubcutaneously.

Preferred vectors include lentivirus vectors and adenoviral vectors, forexample vectors similar to those described in Foxwell et al (2000) AnnRheum Dis 59 Suppl 1, 154-59 or Bondeson et al (2000) J Rheumatol 27(9),2078-2089.

It will be appreciated that it may be desirable to be able to regulatetemporally expression of the polypeptide(s) (for example TRR signallinginhibitor, for example MyD88lpr) in the cell. Thus, it may be desirablethat expression of the polypeptide(s) is directly or indirectly (seebelow) under the control of a promoter that may be regulated, forexample by the concentration of a small molecule that may beadministered to the patient when it is desired to activate or repress(depending upon whether the small molecule effects activation orrepression of the said promoter) expression of the polypeptide. It willbe appreciated that this may be of particular benefit if the expressionconstruct is stable ie capable of expressing the polypeptide (in thepresence of any necessary regulatory molecules) in the said cell for aperiod of at least one week, one, two, three, four, five, six, eightmonths or one or more years. A preferred construct of the invention maycomprise a regulatable promoter. Examples of regulatable promotersinclude those referred to in the following papers: Rivera et al (1999)Proc Natl Acad Sci USA 96(15), 8657-62 (control by rapamycin, an orallybioavailable drug, using two separate adenovirus or adeno-associatedvirus (AAV) vectors, one encoding an inducible human growth hormone(hGH) target gene, and the other a bipartite rapamycin-regulatedtranscription factor); Magari et al (1997) J Clin Invest 100(11),2865-72 (control by rapamycin); Bueler (1999) Biol Chem 380(6), 613-22(review of adeno-associated viral vectors); Bohl et al (1998) Blood92(5), 1512-7 (control by doxycycline in adeno-associated vector);Abruzzese et al (1996) J Mol Med 74(7), 379-92 (reviews inductionfactors e.g., hormones, growth factors, cytokines, cytostatics,irradiation, heat shock and associated responsive elements).Tetracycline—inducible vectors may also be used. These are activated bya relatively—non toxic antibiotic that has been shown to be useful forregulating expression in mammalian cell cultures. Also, steroid-basedinducers may be useful especially since the steroid receptor complexenters the nucleus where the DNA vector must be segregated prior totranscription.

This system may be further improved by regulating the expression at twolevels, for example by using a tissue-selective promoter and a promotercontrolled by an exogenous inducer/repressor, for example a smallmolecule inducer, as discussed above and known to those skilled in theart. Thus, one level of regulation may involve linking the appropriatepolypeptide-encoding gene to an inducible promoter whilst a furtherlevel of regulation entails using a tissue-selective promoter to drivethe gene encoding the requisite inducible transcription factor (whichcontrols expression of the polypeptide (for example MyD88polypeptide)-encoding gene from the inducible promoters. Control mayfurther be improved by cell-type-specific targeting of the geneticconstruct.

It will be appreciated that the expressed protein is preferaby producedat an appropriate level relative to other proteins involved in MyD88 orTRR signalling for optimal functioning.

The methods or constructs of the invention may be evaluated in, forexample, dendritic cells generated in vitro, as known to those skilledin the art, before evaluation in whole animals. The methods described inGB9930616.9, filed on 24 Dec. 1999, may also be used in the evaluationof the methods or constructs of the invention.

The genetic constructs of the invention can be prepared using methodswell known in the art.

The aforementioned therapeutic molecules, for example signallinginhibitor or enhancer, (for example a MyD88 polypeptide, including adominant negative MyD88 mutant polypeptide such as MyD88lpr) orcompound, chimaeric molecule or construct of the invention or aformulation thereof, may be administered by any conventional methodincluding oral and parenteral (eg subcutaneous or intramuscular)injection. The treatment may consist of a single dose or a plurality ofdoses over a period of time. It is preferred that the therapeuticmolecule (for example polypeptide, chimaeric molecule, construct orformulation) is administered by injection, preferably subcutaneousinjection. It will be appreciated that an inducer, for example smallmolecule inducer as discussed above may preferably be administeredorally.

It may be desirable to locally perfuse an area comprising target cellswith the suitable delivery vehicle comprising the genetic construct fora period of time. For example an organ intended for grafting may beperfused ex vivo. Additionally or alternatively, the delivery vehicle orgenetic construct can be injected directly into accessible areascomprising target cells, for example subcutaneously. Methods ofdelivering genetic constructs, for example adenoviral vector constructsto cells of a patient will be well known to those skilled in the art.

In particular, an adoptive therapy protocol may be used or a gene gunmay be used to deliver the construct to dendritic cells, for example inthe skin.

Adoptive therapy protocols are described in Nestle et al (1998) NatureMed. 4, 328-332 and De Bruijn et al (1998) Cancer Res. 58, 724-731.

An adoptive therapy approach may include the steps of (1) obtainingantigen presenting cells or precursors thereof, preferably dendriticcells or precursors thereof, from the patient; (2) contacting saidantigen presenting cells with a signalling inhibitor or enhancer (orcompound identifiable or identified by a screening method of theinvention), and optionally antigen to which an immune response isrequired, or chimaeric molecule or polynucleotide as discussed above, exvivo; and (3) reintroducing the so treated antigen presenting cells intothe patient.

Suitably, the dendritic cells are autologous dendritic cells which arepulsed with polypeptide(s), for example a signalling inhibitor and anantigen. T-cell therapy using autologous dendritic cells pulsed withpeptides from a tumour associated antigen is disclosed in Murphy et al(1996) The Prostate 29, 371-380 and Tjua et al (1997) The Prostate 32,272-278.

In a further embodiment the antigen presenting cells, such as dendriticcells, are contacted with a polynucleotide which encodes the signallinginhibitor or enhancer. The polynucleotide may be any suitablepolynucleotide and it is preferred that it is capable of transducing thedendritic cell thus resulting in respectively activation or inhibitionof antigen presentation by the antigen presenting cell.

Conveniently, the polynucleotide may be comprised in a viralpolynucleotide or virus, as noted above. For example,adenovirus-transduced dendritic cells have been shown to induceantigen-specific antitumour immunity in relation to MUC1 (see Gong et al(1997) Gene Ther. 4, 1023-1028). Similarly, adenovirus-based systems maybe used (see, for example, Wan et al (1997) Hum. Gene Ther. 8,1355-1363); retroviral systems may be used (Specht et al (1997) J. Exp.Med. 186, 1213-1221 and Szabolcs et al (1997) Blood 90, 2160-2167);particle-mediated transfer to dendritic cells may also be used (Tutinget al (1997) Eur. J. Immunol. 27, 2702-2707); and RNA may also be used(Ashley et al (1997) J. Exp. Med. 186, 1177-1182).

The dendritic cells may be derived from the patient (ie autologousdendritic cells) or from a healthy individual or individuals (MHCmatched or mismatched), treated in vitro as indicated above, followed byadoptive therapy, ie introduction of the so-manipulated dendritic cellsin vivo, which may then activate CTL responses. By “healthy individual”we mean that the individual is generally in good health, preferably hasa competent immune system and, more preferably, is not suffering fromany disease which can be readily tested for, and detected.

Thus, the methods of the invention include methods of adoptiveimmunotherapy.

It is preferred if between about 10³ and 10¹¹ APCs are administered tothe patient; more preferably between 10⁶ and 10⁷ APCs. It is preferredthat the APCs are DCs.

The APCs may be administered by any convenient route. It is preferred ifthe APCs are administered intravenously. It is also preferred if theAPCs are administered locally to the site of the disease (such as atumour or local viral or bacterial infection). Conveniently, the APCsare administered into an artery that supplies the site of the disease orthe tissue where the disease is located.

The APCs may be administered subcutaneously so that they mitgrate tolymph nodes.

Preferably, the APCs are DCs.

The cells (or vaccine) may be given to a patient who is being treatedfor the disease by some other method. Thus, although the method oftreatment may be used alone it is desirable to use it as an adjuvanttherapy.

The APCs, such as DCs, or vaccine may be administered before, during orafter the other therapy.

When the disease to be treated is a cancer it is preferable if thecancer has been, is being or will be treated with a conventional therapyor surgery as well as with the method of the invention. Conveniently,depending on the therapy, the cancer is treated by radiotherapy or bychemotherapy.

Cancer antigens to which an immune response may be required aredisclosed in detail above.

When the disease to be treated is an infection by a pathogen it ispreferable if the infection has been, is being or will be treated with aconventional therapy or surgery.

If the patient to be treated has HIV infection it is preferable if themethod of the invention is used as an adjuvant to other treatment, forexample treatment with a reverse transcriptase inhibitor such as AZT or3TC or combination therapy, for example HART (highly active retroviraltherapy).

When the method of the invention is used to treat a solid tumour it ispreferred if the DCs or vaccine are administered as the firstpost-surgery treatment.

When the method of the invention is used to treat leukaemia it ispreferred if the DCs or vaccine are administered after radiotherapy orchemotherapy. It is also preferred if leukaemia patients are alsotreated with the DCs in combination with bone marrow transplantation.

Cancer therapy, for example adoptive immunotherapy may be most effectivein the control or elimination of minimal residual disease rather than inthe reduction of bulk disease. It is conceivable that immunotherapy maytemporarily increase the dimensions of bulk disease due to influx ofcytotoxic T lymphocytes and consequences of tissue drainage. Extent andbulk of disease may be monitored following therapy but not used as aformal endpoint. Patients are followed up in the routine manner in thelong term to ensure that no long term adverse events are manifest.

Further delivery or targeting strategies may include the following.Ballistic compressed air driven DNA/protein coated nanoparticlepenetration (for example using a BioRad device) of cells in culture orin vivo may be used. Constructs for delivery may preferably havecell-type selective promoters.

Whilst it is possible for a therapeutic molecule as described herein,for example a signalling, enhancer or inhibitor or construct ormolecule, to be administered alone, it is preferable to present it as apharmaceutical formulation, together with one or more acceptablecarriers. The carrier(s) must be “acceptable” in the sense of beingcompatible with the therapeutic molecule (which may be a nucleic acid orpolypeptide) and not deleterious to the recipients thereof. Typically,the carriers will be water or saline which will be sterile and pyrogenfree.

Nasal sprays may be useful formulations.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.Such methods include the step of bringing into association the activeingredient (for example, a signalling enhancer or inhibitor, constructor molecule of the invention) with the carrier which constitutes one ormore accessory ingredients. In general the formulations are prepared byuniformly and intimately bringing into association the active ingredientwith liquid carriers or finely divided solid carriers or both, and then,if necessary, shaping the product.

Formulations in accordance with the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets, each containing a predetermined amount of the activeingredient; as a powder or granules; as a solution or a suspension in anaqueous liquid or a non-aqueous liquid; or as an oil-in-water liquidemulsion or a water-in-oil liquid emulsion. The active ingredient mayalso be presented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder (eg povidone, gelatin, hydroxypropylmethyl cellulose), lubricant,inert diluent, preservative, disintegrant (eg sodium starch glycolate,cross-linked povidone, cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Moulded tablets may be made bymoulding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent. The tablets may optionally becoated or scored and may be formulated so as to provide slow orcontrolled release of the active ingredient therein using, for example,hydroxypropylmethylcellulose in varying proportions to provide desiredrelease profile.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavoured basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouth-washes comprising the active ingredient in asuitable liquid carrier.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose or an appropriate fraction thereof, of an activeingredient.

It should be understood that in addition to the ingredients particularlymentioned above the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavouring agents.

It will be appreciated that the therapeutic molecule, for exampleinhibitor or enhancer or molecule or construct of the invention, can bedelivered to the locus by any means appropriate for localisedadministration of a drug. For example, a solution of the said constructcan be injected directly to the site or can be delivered by infusionusing an infusion pump. The construct, for example, also can beincorporated into an implantable device which when placed at the desiredsite, permits the construct to be released into the surrounding locus.

The construct, for example, may be administered via a hydrogel material.The hydrogel is non-inflammatory and biodegradable. Many such materialsnow are known, including those made from natural and synthetic polymers.In a preferred embodiment, the method exploits a hydrogel which isliquid below body temperature but gels to form a shape-retainingsemisolid hydrogel at or near body temperature. Preferred hydrogel arepolymers of ethylene oxide-propylene oxide repeating units. Theproperties of the polymer are dependent on the molecular weight of thepolymer and the relative percentage of polyethylene oxide andpolypropylene oxide in the polymer. Preferred hydrogels contain fromabout 10% to about 80% by weight ethylene oxide and fiom about 20% toabout 90% by weight propylene oxide. A particularly preferred hydrogelcontains about 70% polyethylene oxide and 30% polypropylene oxide.Hydrogels which can be used are available, for example, from BASF Corp.,Parsippany, N.J., under the tradename Pluronic®.

In this embodiment, the hydrogel is cooled to a liquid state and theconstruct, for example, is admixed into the liquid to a concentration ofabout 1 mg nucleic acid per gram of hydrogel. The resulting mixture thenis applied onto the surface to be treated, for example by spraying orpainting during surgery or using a catheter or endoscopic procedures. Asthe polymer warms, it solidifies to form a gel, and the constructdiffuses out of the gel into the surrounding cells over a period of timedefined by the exact composition of the gel.

The construct, for example, can be administered by means of otherimplants that are commercially available or described in the scientificliterature, including liposomes, microcapsules and implantable devices.For example, implants made of biodegradable materials such aspolyanhydrides, polyorthoesters, polylactic acid and polyglycolic acidand copolymers thereof, collagen, and protein polymers, ornon-biodegradable materials such as ethylenevinyl acetate (EVAc),polyvinyl acetate, ethylene vinyl alcohol, and derivatives thereof canbe used to locally deliver the construct. The construct can beincorporated into the material as it is polymerised or solidified, usingmelt or solvent evaporation techniques, or mechanically mixed with thematerial. In one embodiment, the construct (including, for example, anantisense oligonucleotide) are mixed into or applied onto coatings forimplantable devices such as dextran coated silica beads, stents, orcatheters.

The dose of the construct, for example, is dependent on the size of theconstruct and the purpose for which is it administered. In general, therange is calculated based on the surface area of tissue to be treated.The effective dose of construct may be dependent on the size of theconstruct and the delivery vehicle/targeting method used and chemicalcomposition of the oligonucleotide but a suitable dose may be determinedby the skilled person, for example making use of data from the animaland in vitro test systems indicated above.

The construct, for example, may be administered to the patientsystemically for both therapeutic and prophylactic purposes. Theconstruct, for example may be administered by any effective method, asdescribed above, for example, parenterally (eg intravenously,subcutaneously, intramuscularly) or by oral, nasal or other means whichpermit the construct, for example, to access and circulate in thepatient's bloodstream. Construct administered systemically preferablyare given in addition to locally administered constrict, but also haveutility in the absence of local administration.

It is believed that uptake of the nucleic acid and expression of theencoded polypeptide by dendritic cells may be the mechanism of primingof the immune response; however, dendritic cells may not be transfectedbut are still important since they may pick up expressed peptide fromtransfected cells in the tissue.

It is preferred if the vaccine, such as DNA vaccine, is administeredinto the muscle. It is also preferred if the vaccine is administeredonto or into the skin.

A further aspect of the invention provides a vaccine effective againstcancer, or cancer or tumour cells, or against a pathogenic organism orcell infected with a pathogenic organism, comprising an effective amountof a signalling enhancer or activator of antigen presentation as definedabove, or a nucleic acid encoding such a signalling enhancer oractivator of antigen presentation. The vaccine preferably furthercomprises an antigen (or polynucleotide encoding an antigen) having anepitope present on the cancer or tumour cells, or the pathogenicorganism or cell infected with a pathogenic organism.

For example, the invention provides a vaccine effective against cancer,or cancer or tumour cells, or against a pathogenic organism or cellinfected with a pathogenic organism, comprising an effective amount ofMal or TIRAP or TollIP or a fragment, variant or fusion thereof, orpolynucleotide encoding same. The vaccine preferably further comprisingan antigen (or polynucleotide encoding an antigen) having an epitopepresent on the cancer or tumour cells, or the pathogenic organism orcell infected with a pathogenic organism.

It is particularly preferred if the vaccine is a nucleic acid vaccine.Polynucleotide-mediated immunization therapy of cancer is described inConry et al (1996) Seminars in Oncology 23, 135-147; Condon et al (1996)Nature Medicine 2, 1122-1127; Gong et al (1997) Nature Medicine 3,558-561; Zhai et al (1996) J. Immunol. 156, 700-710; Graham et al (1996)Int. J. Cancer 65, 664-670; and Burchell et al (1996) pp 309-313 In:Breast Cancer, Advances in biology and therapeutics, Calvo et al (eds),John Libbey Eurotext, all of which are incorporated herein by reference.

Conveniently, the nucleic acid vaccine may comprise any suitable nucleicacid delivery means, as noted above. The nucleic acid, preferably DNA,may be naked (ie with substantially no other components to beadministered) or it may be delivered in a liposome or as part of a viralvector delivery system.

The nucleic acid vaccine may be administered without adjuvant. Thenucleic acid vaccine may also be administered with an adjuvant such asBCG or alum. Other suitable adjuvants include Aquila's QS21 STIMULON(Aquila Biotech, Worcester, Mass., USA) which is derived from saponin,mycobacterial extracts and synthetic bacterial cell wall mimics, andproprietory adjuvants such as Ribi's DETOX. Quil A, anothersaponin-derived adjuvant, may also be used (Superfos, Denmark). Otheradjuvants such as Freund's may also be useful. It is preferred if thenucleic acid vaccine is administered without adjuvant.

A further aspect of the invention provides a pharmaceutical compositioncomprising a signalling inhibitor or enhancer or binding partner ofMyD88 or MyD88 polypeptide or Mal, TIRAP or TollIP or compoundidentified or identifiable by a screening method of the invention, orpolynucleotide encoding same, or composition or chimaeric molecule,vaccine or polynucleotide of the invention, and a pharmaceuticallyacceptable carrier.

A further aspect of the invention provides a method of killing targetcells in a patient which target cells aberrantly express a firstepitope, the method comprising the steps of (1) obtaining antigenpresenting cells (APCs) from said patient; (2) contacting said APCs witha modulator, preferably an inhibitor, of TRR signalling in dendriticcells or with a polynucleotide or expression vector encoding aninhibitor of TRR signalling in APCs ex vivo; (3) optionally contactingsaid cells with the said epitope or with a polynucleotide or expressionvector encoding the said epitope and (4) reintroducing the so treatedAPCs into the patient.

Upon reintroduction into the patient, the APCs stimulate a cytotoxic Tcell response to the target cells.

Typically the APCs are dendritic cells.

The target cells may be cancer cells.

A further aspect of the invention provides a method of treating apatient with or at risk of cancer or infection with a pathogenicorganism, comprising the step of supplying (1) an modulator, preferablyinhibitor of TRR signalling (which may be Mal or TIRAP or TollIP (or afragment, variant or fusion any thereof), as discussed above; and whichactivates APCs, for example DCs, for example enhances antigenpresentation by APCs, for example DCs) or (2) an inhibitor of asignalling step directly involving MyD88, or (3) a dominant negativemutant of MyD88, to the patient or to an antigen presenting cell, orprecursor cell, of the patient.

A further aspect of the invention provides a method of treating apatient with or at risk of an autoimmune disease or transplantrejection, comprising the step of supplying (1) a modulator, preferablyactivator of TRR signalling (which may be Mal or TIRAP or TollIP or afragment, fusion or variant thereof, as discussed above; and whichinhibits APCs, for example DCs, for example inhibits antigenpresentation by APCs, for example DCs) or (2) an activator of asignalling step directly involving MyD88, or (3) MyD88, to the patientor to a an antigen presenting cell, or precursor cell, of the patient.

A further aspect of the invention provides the use of (1) a modulator,preferably inhibitor of TRR signalling (which may be Mal or TIRAP orTollIP or a fragment, variant or fusion thereof, as discussed above) or(2) an inhibitor of a signalling step directly involving MyD88, or (3) adominant negative mutant of MyD88 (or polynucleotide encoding same) inthe manufacture of a medicament for treating a patient in need ofenhancement of antigen presentation and/or with or at risk of cancer orinfection with a pathogenic organism.

Preferably the medicament further comprises an antigen or polynucleotideencoding an antigen. Still more preferably, the medicament comprises apolynucleotide encoding an antigen and the inhibitor molecule, or achimaeric molecule of the invention.

The medicament may alternatively comprise an appropriate compoundidentifiable or identified by a screening method of the invention.

A further aspect of the invention provides the use of (1) a modulator,preferably activator of TRR signalling (which may Mal or TIRAP or TollIPor a fragment, fusion or variant thereof, as discussed above) or (2) anactivator of a signalling step directly involving MyD88, or (3) MyD88(or polynucleotide encoding same) in the manufacture of a medicament fortreatment of a patient with or at risk of an autoimmune disease ortransplant rejection.

Preferably the medicament further comprises an antigen or polynucleotideencoding an antigen. Still more preferably, the medicament comprises apolynucleotide encoding an antigen and the inhibitor molecule, or achimaeric molecule of the invention.

The medicament may alternatively comprise an appropriate compoundidentifiable or identified by a screening method of the invention.

A further aspect of the invention provides a kit of parts or compositionor chimaeric molecule, comprising (1) Mal or TollIP or TRAP or afragment, derivative or fusion thereof (for which preferences are asindicated above) (or a polynucleotide encoding such a portion) and (2)an antigenic portion comprising or encoding an antigenic molecule. Afurther aspect of the invention provides the said kit of parts orcomposition or chimaeric molecule for use in medicine. A still furtheraspect of the invention provides the use of the said kit of parts or orchimaeric molecule in the manufacture of a medicament for treating apatient in need of modulation of antigen presentation.

Either or both portions in these aspects of the invention may furthercomprise a translocating portion and/or a cell binding portion, asdiscussed above.

The invention is now described by reference to the following,non-limiting, figures and examples.

FIG. 1: Structure of MyD88 comprising a toll and a death domain

MyD88 has a modular organization comprising an N-terminal death domain(DD) separated by a short linker from a C-terminal toll domain. TheN-terminal DD is related to a motif of approximately 90 amino acidsshared between the cytoplasmic tails of the FAS/APO1/CD95 and TNFreceptors and known to mediate protein interactions with other DDsequences forming either homo- or heterodimers (Boldin M. P. et al. JBiol Chem 1995). These interactions form the foundation for buildingsignalling complexes that can activate MAP kinases and the transcriptionfactor NF-κB (Nagata S. Cell 1997). The MyD88 C-terminal toll domaincomprises approximately 130 amino acids that is found in the expandingfamily of Toll-related receptors that comprise the toll-like, IL-1 andIL-18 receptors. Toll domains are also involved in protein-proteininteractions. MyD88 self-associates in yeast. Full-length MyD88 andMyD88 mutants are schematically represented. The black and grayrectangles represent the death and Toll domains, respectively. MyD88containing a point mutation, F56N, in the death domain (represented by awhite circle) is referred to as MyD88-lpr. (Adapted from Burns et al1998 JBC 273 12203).

FIG. 2: Expression pattern of toll-like receptors 1-5 in cells of theimmune system

Recently, Muzio et al. (J Immunol 2000) have examined the expressionpattern (FIG. 2A) of TLR1-5 (FIG. 2B) and found that TLR1 isubiquitously expressed in cells of the immune system, whereas TLR2, 4and 5 are restricted to polymorphonuclear cells, monocytes and dendriticcells. TLR3 seems to be restricted to dendritic cells.

FIG. 3: Dominant negative (inhibitory) MyD88 blocks activation of anNF-κB reporter in 57A HELA cells in response to IL-1

57A HELA cells stably transfected with an NF-κB reporter gene were leftuninfected or infected with a control adenovirus encoding GFP, anirrelevant protein (AdGFP), IκBα (Ad IκBα), wild-type MyD88, the MyD88mutant known as MyD88-lpr or a MyD88-toll mutant only. After one day,cells were stimulated with 20 ng/ml TNF or IL-1 and luciferase activityfrom the NF-κB reporter gene measured. Expression of the dominantnegative (inhibitory) MyD88-lpr protein could specifically inhibit theIL-1 but not TNF-induced NF-κB activation, whereas wild-type MyD88 hadno effect as expected.

FIG. 4: Expression of dominant negative (inhibitor) but not wild-typeMyD88 blocks p38 MAPK, p42/44 MAPK and NF-κB activation as well as IκBαdegradation in human skin fibroblasts (HSF) in response to IL-1

Primary human skin fibroblasts (HSF) were infected in serum-free RPMIwith an adenovirus encoding a control protein β-gal, wild-type MyD88 orthe dominant negative form MyD88-lpr. After 2 h the virus was removedand the cells were cultured in 5% FCS RPMI for 1 day to allow expressionto take place. Then, they were stimulated with 20 ng/ml IL-1 or TNFα for30 mm and cells were lysed for cytosolic and nuclear extracts. Westernblotting was used to analyse cytosolic extracts for MyD88 (FIG. 4A),phosphorylated p38 MAPK (FIG. 4C) and p42/44 MAPK (FIG. 4D), and IκBα(FIG. 4E). Electrophoretic mobility shift assay (EMSA) was used toanalyse nuclear NE-κB-DNA binding activity (FIG. 4B).

FIG. 5: Expression of dominant negative (inhibitor) but not wild-typeMyD88 blocks IL-1 induced IL-6 production in HSF

Primary human skin fibroblasts (HSF) were infected in serum-free RPMIwith an adenovirus encoding a control protein β-gal, wild-type MyD88 orthe dominant negative form MyD88-lpr. After 2 h the virus was removedand the cells were cultured in 5% FCS RPMI for 1 day to allow expressionto take place. Then, they were stimulated with 20 ng/ml IL-1 for 24 h,supernatants collected and analyzed by ELISA for IL-6 production.Expression of the dominant negative MyD88-lpr could inhibit IL-1-inducedIL-6 production in human skin fibroblasts.

FIG. 6: Expression of dominant negative (inhibitor) or wild-type MyD88does not block TNFα-induced IL-6 production in HSF

Primary human skin fibroblasts were infected in serum-free RPMI with anadenovirus encoding a control protein β-gal, wild-type MyD88 or thedominant negative from MyD88-lpr. After 2 h the virus was removed andthe cells were cultured in 5% FCS RPMI for 1 day to allow expression totake place. Then, they were stimulated with 20 ng/ml IL-1 for 24 h,supernatants collected and analyzed by ELISA for IL-8 production.Expression of the dominant negative MyD88-lpr could inhibit IL-1-inducedIL-8 production in human skin fibroblasts.

FIG. 7: Expression of dominant negative (inhibitor) but not wild-typeMyD88 blocks IL-1 induced IL-8 production in HSF

Primary human skin fibroblasts were infected in serum-free RPMI with anadenovirus encoding a control protein β-gal, wild-type MyD88 or thedominant negative form MyD88-lpr. After 2 h the virus was removed andthe cells were cultured in 5% FCS RPMI for 1 day to allow expression totake place. Then, they were stimulated with 20 ng/ml TNFα for 24 h,supernatants collected and analyzed by ELISA for IL-6 production.Expression of the dominant negative MyD88-lpr could not inhibitTNFα-induced IL-6 production in human skin fibroblasts showing thatMyD88 is required specifically for the IL-1 but not the TNF signallingpathway.

FIG. 8: Expression of dominant negative (inhibitor) or wild-type MyD88does not block TNFα-induced IL-8 production in HSF

Primary human skin fibroblasts were infected in serum-free RPMI with anadenovirus encoding a control protein β-gal, wild-type MyD88 or thedominant negative form MyD88-lpr. After 2 h the virus was removed andthe cells were cultured in 5% FCS RPMI for 1 day to allow expression totake place. Then, they were stimulated with 20 ng/ml TNFα for 24 h,supernatants collected and analyzed by ELISA for IL-8 production.Expression of the dominant negative MyD88-lpr did not inhibitTNFα-induced IL-8 production in human skin fibroblasts showing thatMyD88 is required specifically for the IL-1 but not the TNF signallingpathway.

FIG. 9: Expression of dominant negative (inhibitor) but not wild-typeMyD88 blocks p42/44 MAPK and NF-κB activation as well as IκBαdegradation in human umbilical vein endothelial cells (HUVEC) inresponse to IL-1 and LPS

Primary umbilical vein endothelial cells (HUVEC) were infected inserum-free RPMI with an adenovirus encoding a control protein β-gal orthe dominant negative form MyD88-lpr. After 2 h the virus was removedand the cells were cultured in 5% FCS RPMI for 1 day to allow expressionto take place. Then, they were stimulated with 20 ng/ml IL-1 or 1 μg/mILPS for 30 mm and cells were lysed for cytosolic and nuclear extracts.Western blotting was used to analyse cytosolic extracts for MyD88expression (FIG. 9A), phosphorylation of p42/44 MAPK (FIG. 9C), and IκBα(FIG. 9B), expression. Electrophoretic mobility shift assay (EMSA) wasused to analyse nuclear NF-κB-DNA binding activity (FIG. 9D).

FIG. 10: Expression of dominant negative (inhibitor) but not wild-typeMyD88 blocks IL-1- and LPS-induced but not TNF-induced IL-6 productionin HUVEC

Primary HUVEC were infected in serum-free RPMI with an adenovirusencoding a control protein β-gal, wild-type MyD88, the dominant negativeform MyD88-lpr, IκBα or a dominant-negative form of IKK-2. After 2 h thevirus was removed and the cells were cultured in 5% FCS RPMI for 1 dayto allow expression to take place. Then, they were stimulated with 20ng/ml IL-1, 20 ng/ml TNFα or 1 μg/ml LPS for 24 h, supernatantscollected and analyzed by ELISA for IL-6 production. Expression of thedominant negative MyD88-lpr could inhibit IL-1- and LPS-induced IL-6production suggesting that it is a specific inhibitor of toll-relatedreceptors. IκBα and dominant-negative IKK-2 also blocked IL-6-productionas they act downstream of MyD88 in the toll-related receptor signaltransduction pathway. The TNF-induced IL-6 production was not blocked asMyD88 is not involved. However, IKK-2 and IκBα are also utilised by theTNF-R signal transduction pathway.

FIG. 11: Expression of dominant negative (inhibitor) but not wild-typeMyD88 blocks IL-1- and LPS-induced but not TNF-induced IL-8 productionin HUVEC

Primary HUVEC were infected in serum-free RPMI with an adenovirusencoding a control protein β-gal, wild-type MyD88, the dominant negativeform MyD88-lpr, IκBα or a dominant-negative form of IKK-2. After 2 h thevirus was removed and the cells were cultured in 5% FCS RPMI for 1 dayto allow expression to take place. Then, they were stimulated with 20ng/ml IL-1, 20 ng/ml TNFα or 1 μg/ml LPS for 24 h, supernatantscollected and analyzed by ELISA for IL-8 production. Expression of thedominant negative MyD88-lpr could inhibit IL-1- and LPS-induced IL-8production suggesting that it is a specific inhibitor of toll-relatedreceptors. IκBα and dominant-negative IKK-2 also blocked IL-8-productionas they act downstream of MyD88 in the toll-related receptor signaltransduction pathway. The TNF-induced IL-8 production was not blocked asMyD88 is not involved. However, IKK-2 and IκBα are also utilised by theTNF-R signal transduction pathway.

FIG. 12: Expression of dominant negative (inhibitor) MyD88 in dendriticcells has the opposite effect: it induces NF-κB activation

Immature DC were generated from peripheral blood monocytes after 5 daysof culture with 50 ng/ml GM-CSF and 10 ng/ml IL-4. Then, they were leftuninfected, or infected in serum-free medium with a control adenoviruswithout insert (Ad0) and an Adenovirus encoding dominant-negative MyD88(AdMyD88-lpr). A multiplicity of infection of 100 was used. After 6 hand 24 h expression, cells were lysed and their nuclear extractsexamined for NF-κB DNA-binding activity by EMSA. Surprisingly,expression of dominant negative MyD88 could induce on its own NF-κBactivation which is totally in constrast with what has been previouslyfound.

FIG. 13: Expression of dominant negative (inhibitor) but not wild-typeMyD88 induces TNFα production on its own and does not inhibitLPS-induced TNFα production in dendritic cells

Immature DC were generated from peripheral blood monocytes after 5 daysof culture with 50 ng/ml GM-CSF and 10 ng/ml IL-4. Then, they were leftuninfected, or infected in serum-free medium with with a controladenovirus encoding β-gal (Adβ-gal), an adenovirus encodingdominant-negative MyD88 (Adlpr) and an adenovirus encoding wild-typeMyD88 (AdMyD88wt). Adtoll has beenshown to be non-functional and shouldbe ignored. A multiplicity of infection of 100 was used. After 24 h,cells were stimulated with 100 ng/ml LPS. Surprisingly, expression ofdominant negative MyD88 could induce dendritic cell TNFα production onits own, in the absence of additional stimulation. Moreover, it couldnot inhibit LPS-induced TNFα production, a finding that is in constrastwith everything previously found.

FIG. 14: Expression of wild-type MyD88 abrogates theMyD88-lpr(dominant-negative)-induced TNFα production in dendritic cells

Immature DC were generated from peripheral blood monocytes after 5 daysof culture with 50 ng/ml GM-CSF and 10 ng/ml IL-4. Then, they were leftuninfected, or infected in serum-free medium with a control adenovirusencoding β-gal (Adβ-gal), an adenovirus encoding wild-type MyD88(AdMyD88wt) and an adenovirus encoding dominant-negative MyD88 (Adlpr).In some cases, double infection by Adlpr and Adβ-gal or AdMyD88wt atratios 1:1 and 1:5 was used. A multiplicity of infection of 100 was usedfor single infections. After 24 h, cells were stimulated with 100 ng/mlLPS. Surprisingly, expression of dominant negative MyD88 could inducedendritic cell TNFα production on its own, in the absence of additionalstimulation. Moreover, it could not inhibit LPS-induced TNFα production,a finding that is in contrast with everything previously found.

FIG. 15: Expression of dominant-negative MyD88 in dendritic cellsenhances antigen-specific T cell proliferation

Immature DC were generated from peripheral blood monocytes after 5 daysof culture with 50 ng/ml GM-CSF and 10 ng/ml IL-4. Then, they were leftuninfected, or infected in serum-free medium with a control adenoviruswithout insert (Ad0), an adenovirus encoding green fluorescent proteinas a prototype antigen (AdGFP), and an adenovirus encoding GFP linkedtogether with the dominant negative MyD88 (AdGFP-lpr). After 48 h,graded doses of dendritic cells were cultured with 2×10⁴antigen-specific T cells and proliferation was measured at day 3.Delivery of the antigen GFP to dendritic cells induced antigen-specificT cell proliferation that was enhanced by expression of dominantnegative MyD88. This is in agreement with our unexpected result thatinhibition of MyD88 activity in dendritic cells, but not human skinfibroblasts or HUVEC, induces dendritic cell activation.

FIG. 16: Expression of dominant-negative MyD88 in dendritic cellsenhances the allogeneic mixed lymphocyte reaction (MLR)

Immature DC were generated from peripheral blood monocytes after 5 daysof culture with 50 ng/ml GM-CSF and 10 ng/ml IL-4. Then, they were leftuninfected, or infected in serum-free medium with a control adenoviruswithout insert (Ad0), and an adenovirus encoding the dominant negativeform of MyD88 (Adlpr). After 48 h, graded doses of dendritic cells werecultured with 1×10⁵ allogeneic T cells and proliferation was measured atday 6. Expression of dominant negative MyD88 enhances the allogeneic Tcell proliferation, a finding that is indicative of increased DC antigenpresentation.

FIG. 17: Expression of dominant-negative MyD88 in dendritic cellsenhances the expression of costimulatory molecules (CD80, CD86)

Immature DC were generated from peripheral blood monocytes after 5 daysof culture with 50 ng/ml GM-CSF and 10 ng/ml IL-4. Then, they were leftuninfected, or infected in serum-free medium with a control adenovirusencoding GFP, and with an adenovirus encoding dominant negative MyD88(Adlpr). After 48 h, dendritic cells were collected and stained for CD80(FIG. 17A) and CD86 (FIG. 17B), two very important costimulatorymolecules required for efficient antigen-presenting function. Expressionof the dominant negative form of MyD88 enhanced CD80 and CD86 cellsurface expression, which is indicative of enhanced dendritic cellantigen-presenting function.

FIG. 18: Expression of dominant-negative MyD88 in macrophages inducesp38 MAPK phosphorylation

Human macrophages were differentiated from peripheral blood monocytes byaddition of 100 ng/ml M-CSF for 2-3 days in 5% FCS RPMI. Then, they wereleft uninfected, infected in serum-free medium with a control adenovirusencoding β-gal, or infected with an adenovirus encoding dominantnegative MyD88 (Adlpr). An moi of 100 (or 50 in one case) was used asshown. After 6, 24 and 48 h, cells were lysed and extracts assayed forp38 MAPK activity using western blotting and phospho-p38 MAPK-specificantibodies. Unexpectedly, expression of dominant-negative MyD88 inducesp38 MAPK activity in human macrophages.

FIG. 19: Expression of dominant-negative MyD88 in macrophages inducesIRAK phosphorylation

Human macrophages (FIG. 19A) were differentiated from peripheral bloodmonocytes by addition of 100 ng/ml M-CSF for 2-3 days in 5% FCS RPMI.Hela cells (FIG. 19B) cultured in 5% FCS DMEM were also used. Both celltypes were left uninfected, infected in serum-free medium with a controladenovirus encoding β-gal, an adenovirus encoding wild-type MyD88 or anadenovirus encoding dominant negative MyD88 (Adlpr). An moi of 100 wasused. After 5 mm or 12 h, cells were lysed and extracts assayed for IRAKand phospho-IRAK using western blotting. In Hela cells, expression ofdominant negative MyD88 (MyD88-lpr) was found to inhibit IL-1-inducedactivation of IRAK. Unexpectedly, however, expression ofdominant-negative MyD88 induces IRAK activity in human macrophages thatis not increased by the addition of LPS. This finding suggests thatMyD88 activity is also required for an inhibitory signal in macrophages(but not Hela cells) that inhibits IRAK phosphorylation, and itsblockade results in the activation of IRAK.

FIG. 20: Expression of dominant-negative MyD88 in macrophagaes inducesIκBα phosphorylation

Human macrophages were differentiated from peripheral blood monocytes byaddition of 100 ng/ml M-CSF for 2-3 days in 5% FCS RPMI. Cells infectedin serum-free medium with a control adenovirus encoding β-gal or anadenovirus encoding dominant negative MyD88 (Adlpr). An moi of 100 wasused. After 24 h, cells were lysed and extracts assayed for phospho-IκBαusing western blotting. Unexpectedly, expression of dominant-negativeMyD88 induces IκBα phosphorylation in human macrophages.

FIG. 21: Expression of dominant-negative but not wild-type MyD88 inmacrophages induces TNFα, IL-6 and IL-8 production in the absence of anystimulus.

Human macrophages were differentiated from peripheral blood monocytes byaddition of 100 ng/ml M-CSF for 2-3 days in 5% FCS RPMI. Then, they wereleft uninfected, infected in serum-free medium with a control adenovirusencoding β-gal, infected with an adenovirus encoding wild-type MyD88 orinfected with an adenovirus encoding dominant negative MyD88 (Adlpr). Anmoi of 100 was used. FIG. 21A: After 48 h, supernatants were collectedand assayed for TNFα, IL-6 and IL-8 cytokine production in the absenceof any further stimulation. Cytokine production could be detected incells expressing dominant negative MyD88 but not cells expressingwild-type MyD88 or control cells. This suggested that blocking MyD88activity in macrophages, as in dendritic cells but not HSF or HUVEC,results in the activation of cells and the release of inflammatorycytokines. FIG. 21B: At 0 h, 4 h, 24 h and 48 h of expression,supurnatants were collected and assayed by ELISA for TNF, IL-6 and IL-8.Only the results from cells overexpressing dominant negative MyD88(MyD88-lpr) are shown as control cells or cells expressing wild-typeMyD88 had background levels of cytokine production.

EXAMPLE 1 Toll-like Receptor Signalling in Human Cells Materials andMethods

1. Reagents

Human recombinant GM-CSF and TNFα were kind gifts of Dr Glenn Larsen(GI) and Dr D Tracey (BASF), respectively. Human recombinant IL-4 waspurchased from R&D Systems (Minneapolis, USA) and IL-1 was a gift fromHoffman La Roche. LPS was obtained from Sigma Chemical Co. (St Louis,USA).

2. Preparation of Peripheral Blood Mononuclear Cells

Peripheral blood mononuclear cells (PBMC) were obtained by densitycentrifugation of leukopheresis residues from healthy volunteers (NorthLondon Blood Transfusion Service, Colindale, UK). Heparinised residueswere diluted 2× with HBSS and 25 ml were carefully layered over equalvolumes of Ficoll-Hypaque lymphoprep (Nycomed, Oslo, Norway) in 50 mlsterile tubes prior to centrifugation for 30 minutes at 2000 rpm at roomtemperature. After centrifugation, the interface layer was collected andwashed twice with HBSS (centrifuged for 10 minutes at 2000 rpm). PBMCwere then collected and resuspended in 30 ml of RPMI containing 5% FCS.

3. Isolation of Peripheral Blood T Cells and Monocytes

Peripheral blood T cells and monocytes were obtained from PBMC aftercell cell separation in a Beckman JE6 elutriator. Elutriation wasperformed in RPMI containing 1% FCS (elutriation medium). Lymphocyte andmonocyte purity was assessed by flow cytometry usingfluorochrome-conjugated anti-human monoclonal antibodies against CD45,CD3, CD14 and CD19 (Becton Dickinson, Oxford, UK). T lymphocytefractions typically contained ˜80% CD3-expressing cells, ˜6%CD19-expressing cells and <1% CD14-expressing cells. Monocyte fractionsroutinely consisted of >85% CD14-expressing cells, <0.5% CD19 cells and<3% of CD3-expressing cells.

4. Differentiation of Monocytes with M-CSF for Adenoviral Infection

To optimize adenoviral infection, freshly elutriated monocytes werecultured at 1×10⁶ cells/mil in 10 cm petri dishes (Falcon, UK) with 100ng/ml of M-CSF (Genetics Institute, Boston, USA). After 2-3 days theywere washed with PBS to remove non-adherent cells and the remainingadherent monocytes were incubated with 10 ml of cell dissociationsolution (Sigma, UK) for 1 h at 37° C. The cell suspension was washedtwice in RPMI containing 5% FCS and cell viability (90%) was assessed bytrypan blue exclusion. Cells at this stage were 99% CD14 positive byFACS staining and were cultured at 1>10⁶/ml, in 24-well or 48-wellflat-bottomed tissue culture plates (Falcon, UK) for furtherexperiments.

5. Differentiation of Monocytes to Dendritic Cells

Freshly elutriated monocytes were cultured at 1×10⁶ cells/ml in 10 cmpetri dishes (Falcon, UK) in 5% FCS RPMI supplemented with 50 ng/mlGM-CSF and 10 ng/ml IL-4 for 5-6 days. At day 3, cytokines werereplenished. This method presents several advantages as compared todifferentiation of dendritic cells directly from blood or bone marrowprecursors. Besides being easy and giving high numbers of cells, itgenerates a homogenous population of cells with a stable “immature DC”phenotype. This phenotype can be pushed to maturation by addition ofTNFα (10 ng/ml), LPS (100 ng/ml) or monocyte-conditioned medium (50%v/v) for a further 2-3 days to the DC.

6. 57A and Untransfected HELA, HUVEC and HSF

57A HELA cells were a generous gift of R. Hay (University of St Andrews,UK) and were maintained in 5% FCS 1% penicillin/streptomycin DMEM. Humanumbilical vein endothelial cells (HUVEC) were isolated as previouslydescribed (Jaffe E A et al. (1973). J Clin Invest 52: 2745-8) andmaintained in RPMI, in the presence of 10% FCS, 10% NCS, 1%penicillin/streptomycin, 15 μg/ml endothelial growth supplement (Sigma,Poole, UK) and 50 IU/ml heparin. Primary human skin fibroblasts (HSF)were obtained from circumcision speciments cultured in 5% FCS 1%penicillin/streptomycin DMEM in T-75 tissue culture flasks.

8. Adenoviral Vectors and Their Propagation

Recombinant, replication-deficient adenoviral vectors encoding E. coliβ-galactosidase (Adβ-gal) or having no insert (Ad0) were provided by DrsA. Byrnes and M. Wood (Oxford University, UK). The GFP-expressingadenovirus (AdGFP) was generated by double recombination of AdTrack withAdEasy-1 adenoviral plasmid provided by Prof B. Vogelstein (The HowardHuges Medical Institute, Baltimore, USA). AdMyD88wt and AdMyD88lpr weregenerated from plasmids provided by Dr Xu (University of Texas,Southwestern) and Dr K. Burns (Lausanne, Switzerland). In particular,pAdTrackCMV was used for AdMEKK-1 wt, whereas for AdMyD88wt andAdMyD88lpr, a pAdTrack.CMV vector derivative, termed AdTrack.CMVKS17,was used. pAdTrack.CMVKS17 was constructed by removing the EcoRI site ofAdTrack.CMV as well as its multiple cloning site (MCS), and by insertingthe larger multiple cloning site of the vector pBCSK(+) (Stratagene).Recombinant viruses were generated in BJ5183 bacterial cells transformedby the heat-shock method with 1 μg of linearised pAdTrack.CMV-MEKK1 wt,AdTrack.CMVKS17-MyD88wt or AdTrack. CMVKS17-My88lpr constructs and 100ng of replication-deficient adenoviral vector pAdEasy-1. Positiverecombinant clones were selected through their resistance to kanamycin.Following selection, DNA extracted was used for virus propagation in the293 human embryonic kidney cells. Viruses were purified byultracentrifugation through two caesium chloride gradients, as describedin He et al.(He T. C. et al. (1998). Science 281: 1509-12). Titres ofviral stocks were determined by plaque assay, in HEK 293 cells, afterexposure for 1 hour in serum free DMEM medium (Gibco BRL) andsubsequently overlayed with an (1.5%) agarose/(2×DMEM with 4% FCS)mixture (v/v1:1) and incubated for 10-14 days. (He T. C. et al. (1998).Science 281: 1509-12).

9. Adenoviral Infection of Cells

M-CSF-differentiated macrophages and immature or mature dendritic cellswere collected, counted and replated. Then, they were infected inserum-free RPMI with replication-deficient adenoviruses expressing thegene of interest. A multiplicity of infection of 50-100 for macrophagesand immature DC, and 150-300, for mature DC, was used. After 2 h, thevirus was removed and cells were cultured in complete medium for anadditional 1-2 days to allow expression of the protein of interest.Then, they were used in further experiments.

Similarly, 57A HELA, HUVEC and HSF were infected in serum-free mediumfor 2 h. A multiplicity of infection of 40-100 was used. Then, the viruswas removed, complete medium added backed and cells were furthercultured for another 1-2 days.

10. Establishment of Antigen-Specific T Cell Lines

Green fluorescent protein was purchased from Clontech. To establishantigen-specific polyclonal T cell lines, 1×10⁶ PBMC/ml were culturedwith 1 μg/ml green fluorescent protein (antigen) for 7 days and thenwith IL-2 at 20 ng/ml for another 10-14 days. Every 4 days IL-2 wasreplenished. This resulted in the expansion of antigen-specific T cellsand cell death of most other cell populations present in PBMC. After17-21 days of culture, a restimulation step was included by culturingthe T cells with autologous irradiated PBMC at a 1:1 ratio and freshantigen in the absence of IL-2. After 4 days IL-2 was added for another10-14 days. This restimulation cycle was repeated at least 4 timesbefore use of antigen-specific T cells in further experiments.

11. Proliferation Assays of Antigen-Specific T Cells

To assess the specificity of antigen-specific T cell lines, 1×10⁵ ofantigen-specific T cells were cultured with 1×10⁵ autologous irradiatedPBMC and various concentrations of antigen in 96-well flat-bottomedmicrotiter plates. After 3 days, cells were pulsed with [³H]Thymidineovernight and harvested the following day.

To measure dendritic cell-induced antigen-specific T cell proliferation,1×10⁵ antigen-specific T cells were cultured with graded doses ofirradiated or mitomycin-treated dendritic cells that were unplulsed,pulsed with antigen, uninfected or adenovirus-infected. [³H]-Thymidineincorporation was measured after 2 days. All antigen-specificproliferation assays were done in triplicates.

12. Mixed Lymphocyte Reaction (MLR)

To assay the immunostimulatory capacity of non-irradiated or irradiated(3000 rad from a ¹³⁷Cs source) DC, uninfected and adenovirus-infected,DC were cultured in graded doses with 1×10⁵ of allogeneic elutriated Tcells in quadruplicate in a 96-well flat-bottom microtiter plate(Falcon). Proliferation was measured on day 5 by thymidine incorporationafter a 16 h pulse with [³H] thymidine (0.5 μCi/well; Amersham LifeScience, UK).

13. Cytokine Analysis

Cells were infected with adenoviral vectors for 2 h and, then, culturedfor a further 2 days to allow overexpression of the relevant protein tooccur. 24 h after stimulation of the cells, culture supernatants werecollected and kept frozen. Cytokine levels in cell culture supernatantswere measured by standard 2 or 3 layer sandwich ELISA techniques usingspecific monoclonal and polyclonal antibodies for TNFα, IL-4, IL-6,IL-8, IL-12 and IFNγ. Antibody pairs and standards for these assays havebeen purchased from Pharmingen, with the exception of IL-12 reagentsthat were gifted from the Genetics Institute (Boston, USA).

14. Immunofluorescence Staining and Flow Cytometry

For FACS staining, cells were first harvested. For adherent cells wheresurface receptors need to be intact, a warm 2% EDTA in PBS solution wasused for 20 min at 37° C. After cells were in solution, they were washedonce and then resuspended in ice-cold FACS washing buffer. Allsubsequent incubations were performed at 4° C. For each analysis, 5×10⁵cells were incubated with the relevant antigen-specific antibody orisotype control for 30 min and then washed twice with FACS washingbuffer. Cells were then examined by flow cytometry. Cells were ready foranalysis on a FACScan flow cytometer (Becton and Dickinson) by using theCellQuest (Becton Dickinson). Directly conjugated monoclonal antibodiesto HLA-DR, HLA-A,B,C, CD80, CD86, CD3, CD14 and CD25 were purchased byPharmingen, San Diego, USA).

15. Preparation of Cytosolic Protein Extracts

Cytosolic extracts were prepared to investigate biochemical eventsinvolved in signal transduction by western blotting. Adherent cells werescraped from the tissue culture plate/flask into fresh PBS and harvestedby centrifugation (13000 g for 10 seconds at 4° C.). Non-adherent cellswere similarly pelleted by centifugation and washed once with fresh PBS.After discarding the supernatants, an appropriate quantity of ice-coldhypotonic lysis buffer (Whiteside S. T. et al (1992). Nucleic Acids Res20: 1531-8) was added, depending on the number of cells to be lysed(50-100 μl per 1×10⁶ cells). After incubation on ice for 10 minutes,lysates were centrifuged (13000 g, 5 minutes, 4° C.) in order to removenuclei and cell debris. The cleared lysates were then removed to freshtubes, frozen and stored at −20° C. for subsequent estimation of proteinconcentration and use in western blotting.

16. Preparation of Nuclear Protein Extracts

Nuclear protein extracts were prepared to study the NF-κB activation andtranslocation from the cytosol to the nucleus and its DNA-bindingability. After lysis of cells in hypotonic lysis buffer (see section ),nuclei were pelleted by centrifugation (13000 g for 5 minutes at 4° C.),washed once in hypotonic lysis buffer to remove contaminating cytosolicproteins, and then resuspended in hypertonic extraction buffer for 1-2hours at 4° C. under agitation. Hypotonic lysis buffer prevents leachingof proteins out of the nucleus during lysis, whereas hypertonicextraction buffer makes the nuclear membrane porous allowing nuclearproteins to escape into solution. After centrifugation (13000 g for 10minutes at 4° C.) supernatants containing the nuclear protein wereremoved to fresh tubes and stored at −70° C. This method of nuclearextracts preparation is based on that of Whiteside S. T. et al (1992).Nucleic Acids Res 20: 1531-8.

17. Immunoprecipitation

To immunoprecipitate IRAK, cytosolic extracts were incubated with 3 μgof anti-IRAK antibody for 1 h at 4° C. under gentle shaking. Then, 50 μlof 50% slurry protein G sepharose (Amersham) were added and left foranother 2 h shaking. Subsequently, IRAK bound to protein G sepharose wascollected, washed four times, resuspended in Western Blot loadingbuffer, boiled for 5 min and then immediately used for Western blotting.

18. Protein Concentration Assay

Before using cytosolic or nuclear extracts in any further experimentalprocedure (e.g. western blotting, electrophoretic mobility shiftassays), it was necessary to determine their protein concentration inorder to ensure that equivalent amounts of protein were present in eachsample. Protein concentrations were assessed by the Bradford assay.Briefly, 20 μl of appropriately diluted extracts were added intriplicates in a 96-well tissue culture plate along with 20 μl of aseries of BSA concentrations (Sigma, UK) ranging from 10-1000 μg/ml tobe used as a standard. 200 μl of Bradford reagent were then added toeach well, and absorbance was measured at 595 nm in a spectrophotometer(Multiscan Bichromatic, Labsystems). From the linear standard curveformed by the range of BSA concentrations, protein amounts in thecytosolic and nuclear extracts were determined.

19. Western Blotting and Electrophoretic Mobility Shift Assay

Cytosolic proteins were separated by SDS-PAGE on a 10% (w/v)polyacrylamide gel, followed by electrotransfer onto nitrocellulosemembranes. IκBα and IRAK were detected by using antibodies purchasedfrom Santa Cruz Biotechnology (Santa Cruz, USA) and UpstateBiotechnology (USA), respectively, whereas the phosphorylated forms ofIκBα, p38 and p42/44 MAPK were detected by antibodies from New EnglandBiolabs.

20. Luciferase Assay

After LPS stimulation, cells were washed once in PBS and lysed with 100μl of CAT lysis buffer (0.65% (v/v) NP40, 10 mM Tris-HCL pH 8, 0.1 mMEDTA pH8, 150 mM NaCl). Cell lysate (50 μl) was transferred into thewell of a luminometer cuvette strip and Luciferase Assay Buffer (220 μl)added. Luciferase activity was measured with a Labsystem Luminometer bydispensing 30 μl luciferin (1.5 mM, Sigma, Poole, UK) per assay point.Cell lysates were assayed for protein concentration by Bradford assayand the measured luciferase activity was adjusted accordingly.

Results

Myd88 is a cytosolic protein containing toll and death domains (FIG. 1)that has been implicated in the signal transduction mechanisms of TRRmembers TLR (FIG. 2) and IL-1 receptors. A mutein of Myd88. Myd88lpr(FIG. 1), that contains a 53 amino acid deletion at the N-terminus andPhe56Asn mutation, can act as an inhibitor of IL-1, but not TNFactivation of NF-κB (FIGS. 3 and 4) and MAPK activation (FIG. 4) in Helacells (FIG. 3) and human skin fibroblasts (FIG. 4). In agreement withthis result, IL-1 induced production of IL-6 in human skin fibroblastswas also inhibited when Myd88lpr was expressed in these cells (FIG. 5).In contrast Myd88 alone induced IL-6 production without any required forIL-1 activation (FIGS. 5 and 6). In contrast, Myd88lpr had no effect onTNF induced IL-6 production in HSF (FIG. 6). This would be expected asMyd88 is not implicated in TNF signalling. Myd88lpr was also capable ofinhibiting IL-1 (FIG. 7), but not TNF-induced IL-8 (FIG. 8) productionwhen expressed in human skin fibroblasts. The inhibiting effect ofMyd88lpr was not confined to human skin fibroblasts, as studies inHUVECs transfected with AdMyd88lpr showed that IL-1 and LPS-inducedNF-κB activation and p42/44 MAPK activation (FIG. 9) as well as IL-6 andIL-8 production (FIGS. 10 and 11) were also inhibited. In contrast tothese results, we were surprised to observe that, when expressed inimmature DC, Myd88lpr activated NF-κB (FIG. 12) without any requirementfor additional stimulus. Moreover, expression of Myd88lpr was capable ofinducing TNF production by immature DC (FIG. 13) whereas, unlike HSF,Myd88 had no effect (FIG. 13). The activating effect in DC of Myd88lprwas antagonised by Myd88, suggesting that there was competition by thetwo species for the same signalling pathway (FIG. 14). The activatingeffect of Myd88lpr translated to enhanced antigen presenting function ofDC as shown by studies using GFP antigen specific T cells (FIG. 15) orin an allogenic MLR (FIG. 16). The antigen presenting function of DC isassociated with the upreculation of the expression of costimulatorymolecules on the suface of DC, such as CD80 and CD86. In agreement withits stimulating effect, Myd88lpr was found to enhance the expression ofCD80 and CD86 when expressed in immature DC (FIG. 17). The activatingeffect of Myd88lpr was not confined to NT-κB, as expression of Myd88lprin another potential APC, MCSF-human macrophages caused the activationof p38 MAPK (FIG. 18) and IRAK (FIG. 19). In contrast, but in agreementwith the previous studies in Hela cell shown in FIG. 3, Myd88lprinhibited IL-1-induced IRAK phosphorylation (FIG. 19). As for DC,Myd88lpr also induced IκBα phosphorylation in human MCSF macrophages(FIG. 20) as well as inducing or enhancing the production of TNF, IL-6and IL-8 by human macrophages (FIG. 21).

Discussion

Toll related receptors include toll like receptors (TLR) that aretrans-membrane receptor proteins with an extracellular proteincontaining leucine-rich repeats that may recognise LPS-LBP-CD14 complexand a cytoplasmic domain (toll domain) that is also found in theintracellular portion of the IL-1R and related molecules (Gay N J andKeth F H, Rock F et al 1997). TRR commonly utilise signallingcomponents, such as Myd88, IRAK and TRAF6. In particular, Myd88 like theTRR, also contains a toll domain, which, via homotypic interaction,associates with the toll domain of the receptors. Myd88 also has a deathdomain by which it associates with IRAK and thus links the TRR withintracellular signalling pathways. It has been previously shown thatexpression of a dominant negative Myd88 inhibited IL-1 and LPSsignalling in 293 cells (Mujio et al 1998) and both cytokine release andMAPK/NF-κB activation were inhibited. Similar results were obtained inhuman dermal micro-vessel endothelial cells and in the promonocytic cellline, THP-1 (Zhang FX et al 1999) where the transient overexpression ofdominant negative Myd88 blocked IL-1 and LPS-induced NF-κB activation.

Myd88 knock-out mice are unable to induce LPS-dependent TNFα production(Kawai et al 1999). However, although IL-1 and IL-18 induced MAPK andNF-κB activation are knocked out, LPS-induced activation of thesepathways in murine Myd88^(−/−) macrophages appears normal, apart frombeing a bit delayed.

We have examined the role of TRRs in human primary cells by using anefficient adenoviral gene transfer technique to over-express wild type(Myd88wt) or a Myd88 mutein, Myd88lpr (FIG. 1). As expected fromprevious studies by Burns et al (1998), Myd88lpr was able to block IL-1signalling in HSF, HUVECs and Hela cells. In addition Myd88lpr blockedLPS signalling in HUVECs. The inhibiting effect of Myd88lpr includedboth the activation of signalling molecules, such as NF-κB and MAPK, aswell as cytokine production. As expected, TNF signalling pathways wereunaffected. Moreover, in HSF, Myd88wt was able to induce cytokineproduction without any additional stimulus. Unexpectedly, however, wefound that the introduction of Myd88lpr into immature DC and MCSFmacrophages, resulted in the activation of NF-κB, MAPK and cytokineproduction. Moreover, in DC, the expression of the costimulatorymolecules CD80 and CD86 centrally involved in the antigen presentingfunction of these APC was upregulated by Myd88 lpr. As a result of theseeffects, the antigen presenting function of DC was greatly enhanced bythe expression of Myd88lpr.

The data would imply that, in DC and macrophages the inhibition of TRRsignalling can result in the activation of the cell. This would suggestthat there may be TRR whose role is to inhibit APC, particularly DCfunction. This suggestion is in-part supported by the observation thatthe stimulating effect of Myd88lpr is inhibited by simultaneousexpression of Myd88wt. Moreover, unlike in Hela cells, where Myd88lprinhibits IRAK activation, in macrophages, Myd88lpr induces theactivation of this kinase.

Besides adding a new layer of complexity to our understanding of TRRsignalling and what roles these receptors may have regulating cellfunction, these data have uncovered a novel pathway involved incontrolling antigen presentation activity. This pathway could beharnessed for the therapeutic modulation of the immune system, both forthe prevention and treatment of autoimmune disease and allergy, and alsoin circumstances where one wishes to stimulate the immune system, suchas in vaccination. The discovery of these pathways may have a profoundimpact on vaccine design and on immuno-modulation approaches in general.

Numbered References

-   1. Rock et al (1998) PNAS 95, 588-593-   2. O'Neill & Dinarello (2000) Immunol Today 21, 206-209-   3. Poltorak et al (1998) Science 282, 2085-2088-   4. Underhill et al (1999) Nature 401, 811-815-   5. Burns et al (1998) J Biol Chem 273, 12203-12209-   6. Kawai et al (1999) Immunity 11, 115-122-   7. Takeuchi et al (2000) Int Immunol 163, 978-984-   8. Du et al (2000) Eur Cytokine Netw 11, 362-371

EXAMPLE 2 Dendritic Cell Culture

Exemplary Dendritic Cell Culture from Normal Volunteers

CD14⁺ peripheral blood monocytes are adhered to tissue culture flasksand cultured in the presence of 1% AB serum, GM-CSF (400 ng/ml) and IL-4(400 IU/ml) for 7 days. This yields cells with the morphology of DC anda mean of 49% with the CD1a⁺ marker which is indicative of the immatureform of the DC capable of taking up and presenting antigen. These cellsare then matured to CD83⁺ cells by the addition of TNFα (15 ng/ml),which enables the DC to present antigen to cytotoxic T-cells. 7% of thecells become CD83⁺ within 1 day, but 3 days at least are required formaximum effect. It is possible that monocyte conditioned medium couldreplace the 1% AB serum but this is probably not desirable.

Exemplary Dendritic Cell Culture from Patients with Cancer

DC are generated from 6 patients with relapsed metastatic disease, bothprior to and following salvage chemotherapy (a total of 12 samples ofperipheral blood, each of 50 mls).

Clinical Study

Patients donate a single unit of autologous blood according to standardprotocol. Patients are evaluated prior to donation by a bloodtransfusion physician. Autologous donations are screened in the same wayas allogeneic donations for routine virus markers (HIV, HBV, HCV andsyphilis) and patients give consent to this after appropriatecounselling if they wish to participate. This precaution protectsclinical and laboratory staff from potential infection and the routineblood supply from the possibility of cross-contamination. The blood istaken into a routine quad-pack. This allows automated separation of redcells, buffy coat and plasma. The buffy coats yields approximately670×10⁶ mononuclear leukocytes which give approximately 47×10⁶ DC usingcurrent techniques. A dosage range of 8-128×10⁶ DC per patient is used.Peripheral blood monocytes are divided into 2 aliquots and pulsed with aTRR signalling inhibitor (for example MyD88lpr, optionally with apromoter of cell uptake) and antigenic peptide, or tumour cell extractbetween days 1 and 10. Alternatively, peripheral blood monocytes are isexposed to a DNA vaccine construct, for example an adenovirus construct,encoding a TRR signalling inhibitor, (for example MyD88lpr) and encodingan antigenic peptide, and cultured for 10 days possibly with multipleexposures to the vaccine. Serum-free culture conditions or autologousplasma is used in preference to allogeneic AB serum. Cultured DCs arepooled, washed and resuspended in 100 mls saline prior to infusion over1 hour. The autologous red cell concentrate is not returned to thepatient other than for a standard clinical indication. The ex vivo DCculture procedures are carried out following good manufacturingpractices.

Patients who donated the initial blood samples will, by this time, havereceived salvage chemotherapy and may or may not be in clinicalremission. Further patients with relapsed metastatic disease receivetreatment prior to receiving chemotherapy. There are two treatmentregimes:

-   -   (1) metastatic relapse, standard therapy followed by adoptive        immunotherapy;    -   (2) metastatic relapse, adoptive immunotherapy followed by        standard therapy.

Product infusion is carried out under the direct supervision of anexperienced physician on a ward on day bed unit where resuscitation andsupportive care facilities are available if required.

1. A method of enhancing presentation of an antigen by a mature orimmature dendritic cell, comprising supplying ex vivo to said cell aninhibitor of Toll-related receptor (TRR) signaling which comprises afunctional Toll domain and does not comprise a functional death domain,wherein the inhibitor is a dominant negative mutant of MyD88; therebyenhancing antigen presentation.
 2. The method of claim 1 wherein theinhibitor of TRR signaling modulates binding of an intracellularmolecule to a TRR.
 3. The method of claim 1 wherein the inhibitor of TRRsignaling modulates binding of MyD88 to a TRR.
 4. The method of claim 1wherein the inhibitor of TRR signaling modulates binding of MyD88 to apolypeptide comprising a death domain (DD).
 5. The method of claim 1wherein the dominant negative mutant is MyD88lpr.
 6. The method of claim1 wherein the dominant negative mutant is expressed in the cell.
 7. Themethod of claim 6 wherein the cell is administered a polynucleotidecapable of expressing the dominant negative mutant in the cell.
 8. Themethod of claim 7 wherein the polynucleotide is administered in anadenovirus vector.
 9. The method of claim 1 further comprising supplyingto said cell said antigen to be presented or a nucleic acid capable ofexpressing said antigen in the cell.
 10. The method of claim 9 whereinboth the inhibitor and the antigen are supplied to the cell byexpression in the cell.
 11. The method of claim 10 wherein the inhibitorand the antigen are provided in a single recombinant polynucleotidemolecule.
 12. A method of enhancing presentation of an antigen by amature or immature dendritic cell, comprising supplying ex vivo to saidcell an inhibitor of Toll-related receptor (TRR) signaling whichcomprises a functional Toll domain, does not comprise a functional deathdomain, and is capable of inhibiting binding of MyD88 to a TRR, whereinthe inhibitor is a dominant negative mutant of MyD88; thereby enhancingantigen presentation.
 13. A method of enhancing presentation of anantigen by a mature or immature dendritic cell, comprising supplying exvivo to said cell a dominant negative mutant of MyD88 comprising afunctional Toll domain and not a functional death domain, therebyenhancing antigen presentation.
 14. A method of enhancing presentationof an antigen by a mature or immature dendritic cell, comprising: (a)supplying ex vivo to said cell a dominant negative mutant of MyD88 and(b) supplying ex vivo to said cell said antigen to be presented or anucleic acid capable of expressing said antigen in the cell; therebyenhancing antigen presentation.
 15. A method of enhancing presentationof an antigen by a mature or immature dendritic cell, comprising: (a)supplying ex vivo to said cell MyD88lpr and (b) supplying ex vivo tosaid cell said antigen to be presented or a nucleic acid capable ofexpressing said antigen in the cell; thereby enhancing antigenpresentation.
 16. A method of enhancing presentation of an antigen by amature or immature dendritic cell, comprising: (a) supplying ex vivo tosaid cell a dominant negative MyD88 and (b) supplying ex vivo to saidcell said antigen to be presented or a nucleic acid capable ofexpressing said antigen in the cell; thereby enhancing antigenpresentation.
 17. The method of claim 14, wherein the dominant negativemutant is expressed in the cell.
 18. The method of claim 17, wherein thecell is administered a polynucleotide capable of expressing the dominantnegative mutant in the cell.
 19. The method of claim 18, wherein thepolynucleotide is administered in an adenovirus vector.
 20. The methodof claim 14, wherein both the inhibitor and the antigen are supplied tothe cell by expression in the cell.
 21. The method of claim 20, whereinthe inhibitor and the antigen are provided in a single recombinantpolynucleotide molecule.